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Deep Learning Technology: Sebastian Arnold, Betty van Aken, Paul Grundmann, Felix A. Gers and Alexander Löser. Learning Contextualized Document Representations for Healthcare Answer Retrieval. The Web Conference 2020 (WWW'20)
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No data is available regarding the pathogenesis of PCV-3 infection. The lack of virus isolation has impeded the establishment of an infection model to date. It is known that PCV-3 can be found in different tissues of domestic pig and wild boar (86, 87, 95), indicating the systemic nature of the infection. However, the point of viral entry, primary replication, organic distribution and persistence are still unsolved issues. PCV-3 has been found in feces, nasal swabs, oral fluids, and trucks transporting pigs (82, 85, 95), which allows speculating that horizontal transmission through direct contact is probably an important route. Detection of viral genome in fetuses and stillborn from farms with history of reproductive failure (21, 70, 75), as well as in semen and colostrum, points out also to vertical transmission as another likely route. Definitively, more studies are needed to ascertain the potential excretion routes of this virus.
One study performed in samples from captured and re-captured wild boar revealed long-lasting infection (potential persistent infection), since the virus was detected during a period of at least 5–7 months in few animals (95). Susceptibility of wild boar to PCV-3 was not a surprise, since this species shows susceptibility to several pathogens that affect humans and animals (123), including PCV-2; moreover, the wild boar can also develop PCV-2-SD (124). Taking into account the potential long period of infection observed in some animals and even a higher overall prevalence in wild boar when compared with domestic pigs, such potential reservoir role deserves further investigations (95, 96).
Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiologic agent that causes porcine reproductive and respiratory syndrome (PRRS), which has been acknowledged as one of the most economically devastating diseases in the swine industry. Typical clinical symptoms of PRRS are characterized as blue-ear in the infected pigs, with mild to severe respiratory disease in newborn/weaned piglets and growing pigs, as well as reproductive failure in pregnant pigs. Furthermore, highly pathogenic PRRSV (HP PRRSV) strains are also associated with high fever, respiratory and reproductive failure, pulmonary lesions, and abnormal host immune response.
The co-infection of swine with PRRSV and PCV2 is common in clinical conditions and contributes to a range of polymicrobial disease syndromes. The co-infection rate of PRRSV and PCV2 has been reported as 42% and up to 85.4% in lungs with proliferative and necrotizing pneumonia lesions in postweaned pigs. A Chinese group reported a co-infection rate of PRRSV and PCV2 of 52.4% in 103 clinical specimens collected from the swine farms of Guizhou province, China. Furthermore, PRRSV- and PCV2-naturally co-infected boars have been found.
Both PCV2 and PRRSV can target host immune cells and impair host defenses, resulting in increased susceptibility to infections by primary and secondary pathogens that can affect growth and performance as well as increase morbidity and mortality. Harms and colleagues found that PCV2 and PRRSV co-inoculated pigs had severe dyspnea, lethargy, and occasional icterus by 10 days post-inoculation (dpi) and were dead by 20 dpi, while PCV2-infected pigs developed lethargy, sporadic icterus and exudative epidermitis with mortality rate of 26%, and PRRSV-inoculated pigs only showed dyspnea and mild lethargy. Moreover, PCV2 and PRRSV co-inoculated pigs also had severe proliferative interstitial pneumonia and hepatic lesions, whereas PRRSV-inoculated pigs only had moderate proliferative interstitial pneumonia without bronchiolar or hepatic lesions or lymphoid depletion. Another study demonstrated that replications of PRRSV and PCV2 were enhanced and that more severe clinical signs and lesions were observed in piglets infected with HP-PRRSV followed by PCV2 infection. During the co-infection, PRRSV influences the infection dynamics of PCV2 subtypes PCV2a and PCV2b by lengthening PCV2 viremia and shedding in vivo, while PCV2 infection increases the rate of amino acid mutations of PRRSV during serial passages in pigs. In addition, the mutation rates in ORF5 and ORF6 of PRRSV were significantly higher in concurrently PRRSV/PCV2b co-infected pigs than that of pigs infected with PRRSV only, while a significantly higher mutation rate in ORF7 of PRRSV was detected in the PRRSV/PCV2a co-infected pigs.
Moreover, it has been reported that the co-infection of pigs with PRRSV and PCV2 results in an increased expression profile of IL-1β and TLR2, 4 and 8, and it has a negatively synergistic effect on the mRNA expression of TLRs 3, 7 and 9, A20, Bcl-3, IRAK-M, MKP-1, SARM1 and SIGIRR, as well as IRF-1, IRF-3, IFN-α and TNF-α. In contrast, co-infection induces significantly lower levels of anti-PCV2 and anti-PRRSV IgG antibodies. Furthermore, PCV2/PRRSV co-infection plays an immunomodulatory role in the pathogenesis of PCVAD by dramatically decreasing the total and differential leukocyte counts and inducing significantly higher numbers of T(regs) in dendritic cells (DCs). Thereafter, the levels of the inhibitory markers PD-L1 and IL-10 are significantly increased, while the level of the stimulatory markers CD86 is significantly decreased. To date, effective treatment for PRRSV infection is not available, partly because co-infection with PCV2 and PRRSV may interfere with the vaccine. It has been reported that PCV2 vaccination is effective at inducing a neutralizing antibody response and significantly reduces PCV2-associated lesions and viremia in pigs co-infected with PCV2 and PRRSV. However, vaccination with a modified live PRRSV vaccine followed by challenge with PRRSV and PCV2 protected against PRRS but enhanced PCV2 replication and pathogenesis. These results indicate that synergistic effects occur during PRRSV and PCV2 co-infection, as the clinical signs and lesions in the co-infected pigs are more severe than that of PCV2 or PRRSV singularly infected pigs. Furthermore, mutation rates of PRRSV may increase during PRRSV/PCV2 co-infection.
Transmission of viruses from wildlife to humans continues to cause outbreaks of disease in humans. Examples of recent outbreaks are the Middle East Respiratory Syndrome-coronavirus (MERS-CoV) that may have originated from bats and/or camelids and the influenza A (H7N9) virus that originated from wild birds. A systematic exploration of viruses present in several key host species of wild animals might provide important information to find the original host or carriers of viruses of future outbreaks of viral disease among domestic animals, endangered animal species, and humans. Furthermore, information about the presence of viruses in healthy hosts provides a baseline level for viruses present in these animals in case an outbreak of disease occurs. In previous viral metagenomics studies, high numbers of new viruses have been identified. The results of these studies have highlighted that our knowledge of the viral reservoir is far from complete and many, as yet, unidentified viruses circulate among humans and wild and domestic animals. However, there is an enormous diversity of viral sequences and viral metagenomics efforts should be focused on outbreaks of disease and viral metagenomics on samples collected from a selected number of key species.
Wild carnivores are known carriers of several viral pathogens that can affect domestic animals and humans, including rabies and canine distemper virus. In addition, in previous studies various previously unknown viruses have been detected in European badgers, red foxes and European pine martens in the Netherlands. In the present study, we evaluated the viral diversity of fecal swabs or fecal specimens collected from 10 different small carnivore species of the Mustelidae, Canidae, Viverridae and Felidae families inhabiting northern Spain.
Many groups believe that PCV2 alone is not enough to induce PCVD/PCVAD. However, PCV2 targets lymphoid tissues and strongly impacts T-cell selection processes in the thymus, resulting in an obvious lymphoid depletion and immunosuppression in the pig. Consequently, PCV2 infection results in an increased susceptibility to opportunistic infections of viruses and bacteria. Obviously, co-infections of PCV2 with other viruses may increase pathogenicity in pigs, resulting in more severe clinical symptoms. In addition to dual infections with PCV2 and other viruses, multiple infections are often detected in the field. Moreover, many lines of evidence have shown that bacterial infection, vaccination failure, stress or crowding together with PCV2 can also lead to PCVD/PCVAD. In addition, some PCV2-infected pigs can develop severe diseases; however, PCV2 also evokes a subclinical infection in pigs without any obvious symptoms in many cases. Therefore, PCV2 increases clinical signs of PRRSV, CSFV, SwIV, PRV or PEDV infections, and meanwhile, secondary infection (such as PPV infection) and worse physical/growth condition can also provide a better in vivo environment for PCV2 infection. Thus, more research is needed to improve our understanding of the interactions between different swine viruses and bacteria during co-infection with PCV2 in pigs, including how they interact with the host immune response and how they affect the efficacy of vaccination. These studies could lead to important breakthroughs in the understanding of PCVD/PCVAD and in the development of new strategies to control the disease.
A larger number of AstVs were detected in both rodent and shrew samples (Additional file 1: Table S4). Fifty-five AstVs were selected for sequencing. Most of the rodent AstVs sequenced belonged to four main genetic lineages 1 to 4 within the genus Mamastrovirus and had less sequence similarity with AstVs in other hosts (Fig. 5c). One rodent AstV, RtRn-AstV-1/GD2015, was closely related to AstVs of cattle, deer, and pigs with > 90% nt identity. Two shrew AstVs, Shrew-AstV/SAX2015 and Shrew-AstV/GX2016, were related to mouse AstV with ~ 70% nt identity in the genus Mamastrovirus. Lineage 5 contained one shrew AstV and one mouse AstV, with 79% nt identity with each other. Lineage 5 branched out of the genus Mamastrovirus and showed a closer relationship with the genus Avastrovius.
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
Sixty rodent samples were identified as PicoV positive, and 23 strains underwent genome sequencing (Additional file 1: Table S4). Rodent viruses from the genera Enterovirus, Hunnivirus, Mosavirus, Cardiovirus, Rosavirus, Kobuvirus, and Parechovirus were found in this study and showed 48.3–56.4%, 80.4–80.8%, 47%, 46.8–60.3%, 60.9%, 63–76.9%, and 43.7–87.3% RdRp aa identities with known members in each genus, respectively (Fig. 5b and Additional file 1: Table S11). Eight viruses formed lineages 1 and 2 close to the bat PicoV clade with 38.1–43.6%, 33.5–38.8%, and 48.2–56.7% aa identities with bat PicoVs in the P1, P2, and P3 regions, respectively. Two novel lineages 3 and 4 were identified with < 10.2–28.9% aa identities in the P1 region, 17.3–23.6% in the P2 region, and 21.8–28.4% in the P3 region compared with other PicoVs (Additional file 1: Table S10). Viruses closely related to known PicoVs of other hosts were found (e.g., rodent viruses related to human aichivirus, human rosavirus, and bovine hunnivirus).
Respiratory problems are common in pig herds worldwide and can be associated with significant production losses. They often have a multifactorial background and can be associated with a number of different factors such as environment, management, production system, animal genetics, etc., in addition to different pathogens. Major viral pathogens associated with respiratory disease are PRRSV, PRV, and swine AIV, which are all known to induce respiratory disease and lesions. Other viruses such as paramyxovirus (PMV), porcine cytomegalovirus (PCMV), PCV2, PRCV, and TTSuV are considered minor pathogens that could play a role through co-infection or in combination with other outer factors. In this study, none of the viruses considered major pathogens were detected, which was partly expected, as Sweden has been declared free from PRRSV and PRV. Viruses of minor relevance (TTSuV1 and PCV2) were identified, but only in one pig (R8). In contrast, five SPF pigs were positive for TTSuV1, one of which was also co-infected with TTSuV2.
In general, there was no major difference regarding viruses detected in the two different groups, but the variation was at the individual level. The viruses that differed between the two groups were porcine lymphotrophic herpes virus 1, adeno-associated virus, unclassified circovirus, and porcine sapelovirus A and sapovirus, which were only present in conventionally reared pigs, while TTSuV2 only was detected in one SPF pig. The fact that many of the viruses were found in both groups was not surprising, as many studies have shown a high co-infection rate of several viruses in both healthy pigs and in pigs with different disease complexes. Many respiratory viruses are also known to be ubiquitous in pig populations. It is possible that viral load could play a role in the development of clinical signs, but also that co-infections could have a synergistic effect. For example, enhanced respiratory disease development has been seen in co-infection situations with PRRSV and viruses such as PRCV, swine AIV, and PCV2. The exact mechanisms behind this are not always clear, but it is known that suppression of the immune system and alteration of cytokine responses can be important. Some viruses can also affect macrophage function. Considering that different viruses affect the host in different ways, the order that viruses infect their hosts could be important for particular outcomes, hence making them very complicated to study. Furthermore, bacteria are also known to induce respiratory disease, either alone or through viral-bacterial/bacterial-bacterial interactions. In addition, migration of parasites through the lungs may also aggravate signs of respiratory diseases.
Looking at the specific viruses, it was clear that viruses of certain families were well represented in most of the pigs investigated. For example, adenoviruses were present in all pigs, and 16 out of 18 pigs were positive for picornaviruses. Porcine adenovirus is ubiquitous throughout the world; although the virus has been isolated in connection to disease investigations, it is not considered a major pathogen but rather is believed to often result in subclinical infection. There are three species of porcine adenovirus recognised: porcine mastadenovirus A (porcine adenovirus 1–3), porcine mastadenovirus B (porcine adenovirus 4), and porcine mastadenovirus C (porcine adenovirus 5). Of these, porcine adenovirus 3 and 4 were identified in the different samples from this study. A total of 94% of the pigs were positive for porcine adenovirus 3 and 63% for porcine adenovirus 4. No sequence reads were classified as porcine adenovirus 5. There is a lack of complete genomes of adenoviruses available, making the classification of the reads in this kind of dataset difficult. It is likely, for example, that many of the reads referred to as “other mastadenovirus” in Table 1 could be porcine adenovirus 4 but have been classified as “other mastadenovirus” because only the fibre, pVIII, E1B 55K, and DNA repeat region have been previously sequenced. The dataset may also contain more divergent porcine adenovirus/es that are not classified in any of the present genera.
Different parvoviruses are also known to be present, sometimes at high prevalence, in pigs worldwide. We have previously, using metagenomics, discovered porcine bocavirus 1 in Swedish pigs; PBoV3 has also been shown to be present in Sweden. The pathogenicity of these viruses is not well known, and they have been detected in both healthy pigs and in pigs suffering from both respiratory and enteric diseases. In our study, there was no major difference in the detection rate of these viruses between the two groups. In four of the samples, an additional parvovirus, porcine parvovirus 7, was identified. This virus was first detected in 2016 from a rectal swab collected from an adult pig in the US and showed a very low identity to any known parvovirus. The virus in that study was detected in different sample types and at a detection rate of 8.6%. Since that first discovery, this virus has also been identified in China. The non-structural protein of the PPV7 characterised in Sweden showed a high degree of similarity (93.1–94.6%) to the US and China isolates, while the capsid was more divergent, with a nucleotide identity of 84.7–86.8%. The potential role of this virus in connection to disease has yet to be determined.
Picornaviruses were also detected to a high degree in the investigated pigs, and altogether four different picornaviruses were identified: pasivirus A, posavirus 1, teschovirus, and porcine sapelovirus A. Picornaviruses can cause subclinical infection, as well as diseases ranging from those with mild symptoms such as fever to more severe diseases. In two of the conventionally reared pigs, a high number of teschoviral reads were identified, enabling the assembly of two complete genomes. Porcine teschovirus is endemic in pigs worldwide, and there are 13 known serotypes. Often the infection is subclinical, but PTV-1 is associated with encephalomyelitis (Teschen disease). PTV2, PTV3, and PTV5 have been associated with a milder version called Talfan disease. The viruses identified in these two pigs showed were most similar to PTV-10, which has not been associated with disease, and whether they belong to the serotype 10 remains to be determined.
In some instances, the number of reads that matched to a specific virus was very low, sometimes less than 10 reads (marked with a lighter green in Table 1). It should be noted that although these low read numbers could be due to low abundance viruses or amplification bias, other reasons such as contamination within the sequencing run cannot be ruled out.
In conclusion, we observed a variable co-infection rate in the individual pigs in the two studied groups. The difference was seen on an individual level rather than on a group level. Thus, no specific virus could explain the respiratory disease of these pigs, but the results obtained provide important information on the viruses circulating in pig populations.
Using random amplification in combination with next-generation sequencing, more than 320,000 trimmed sequence reads were obtained of fecal samples collected from the carnivores of the present study (Figure 1). Reads were classified into eukaryotic viruses, phages, bacteria and eukaryotes. Many of the identified sequences were of bacterial or eukaryotic origin. A substantial proportion of the reads did not have any significant hits for nucleotide or amino acid sequences in GenBank. In addition, several reads were detected that had the closest similarity to viruses. In the majority of the samples, sequences of the order Caudovirales were detected and in 26 out of 42 samples, sequences were detected that had the closest similarity to viruses known to infect eukaryotes (Figure 2A, Table 1). Viruses belonging to the families of Anelloviridae, Astroviridae, Bunyaviridae, Caliciviridae, Circoviridiae, Parvoviridae subfamily Parvovirinae, Picobirnaviridae, Picornaviridae, Rhabdoviridae, and Retroviridae were detected (Figure 2B). Furthermore, sequences were detected that had the closest similarity to the recently proposed family of Breviviridae and the recently described hybrid DNA virus NIH-CQV/PHV which was identified as a contaminant of silica column-based nucleic acid extraction kits. No sequences were detected that were identical to currently known zoonotic viruses. A proportion of the detected viral sequences had the closest similarity to viruses previously detected in birds and rodents. For example, in an European mink (sample 26), sequences were detected with >95% homology on the nucleotide level with Turkey parvovirus and in a stone marten (sample 41), sequences were detected with 94-96% homology on the nucleotide level with Encephalomyocarditis virus type 2 isolate RD 1338 (D28/05) detected in a wood mouse (Apodemus sylvaticus). These viruses most likely originate from the diet of the animals. In addition, sequences with >95% identity on the nucleotide level to viruses that are known to infect mink were detected in European and American mink, including Mink calicivirus strain MCV-DL/2007/CN (samples 1 and 8) and Aleutian mink disease virus (sample 30). Antibodies to Aleutian mink disease parvovirus have been detected in a cohort of free-ranging European mink in southwestern France and northern Spain previously, but not in another cohort of free-ranging European mink in Navarra, Spain. Additional sampling and confirmation by specific PCR is necessary to indeed confirm that the Aleutian mink disease parvovirus is circulating among these animals. Besides these sequences that had high homology with known viruses, also sequences were detected that had the closest similarity to viruses, but with only low homology. A number of sequences of potentially novel viruses or virus variants, including a theilovirus, phleboviruses, an amdovirus, a kobuvirus and picobirnaviruses, were further characterized in the present manuscript, while sequences of the other viruses are preliminary and need further characterization.
In this study, we examined the virus diversity in serum samples from Nicaraguan children with unknown acute febrile illness. We performed Virochip microarray and deep sequencing analyses on 7 positive control and 123 undiagnosed samples. Both of these methods succeeded in detecting the expected virus in the positive control samples. Virochip analysis produced putative viral hits in 10/123 (8%) of the previously negative samples, whereas deep sequencing revealed virus or virus-like sequences in 45/123 (37%). This study demonstrates the utility of these metagenomic strategies to detect virus sequence in multiple human serum samples and is the first to utilize second-generation sequencing to simultaneously investigate many cases of acute unknown tropical illness.
Monitoring the emergence and spread of novel human pathogens in tropical regions is a central public health concern. Metagenomic analysis enables more systemic viral detection of both known and novel viral pathogens and can be employed as diagnostic supplements to pathogen detection as part of public health monitoring systems and epidemiologic surveys–,–,,,. Despite the headway, metagenomic virus detection studies will have to confront several remaining difficulties concerning diagnostic accuracy. Foremost concerns include enhancing the sensitivity and specificity of deep sequencing-based diagnostic methods and re-evaluating the evidence for disease causality in light of increasingly sensitive nucleic acid detection and pathogen discovery methods. The former will require improved strategies to biochemically enrich and computationally identify viral sequences while reducing host background sequences. The latter will require a cautious reconsideration of criteria used to establish causal links between microbes and disease, as well as extensive case-by-case follow-up studies employing classical laboratory methods, such as serological analysis and cell culture amplification. It is important to highlight that observing viral sequence in sequencing data is insufficient to establish the role of a virus in disease causality. Like other detection strategies, deep sequencing will serve to inform secondary tests, including seroconversion assays, further nucleic acid testing, cell culture amplification, and additional investigations into plausible disease mechanisms.
We detected virus sequence at concentrations as low as ∼2 in 106 reads. Virus sequence detected in a clinical sample at vanishingly low copy numbers may reflect several possible host-microbe scenarios. The sequence detected may be that of a pathogenic virus capable of causing illness at low copy number or through indirect effects, a ubiquitous non-disease causing microbe, a virus outside of its primary replication site, low-level contamination, an artifact of sample collection timing/processing, or remains of incomplete immune clearance. Additional evidence must be considered in each case to define the host-microbe relationship.
In this study, we compared the performance of the Virochip and deep sequencing for detecting virus sequence in human serum. The limit of detection of the Virochip was approximately one part in 105 for the poliovirus controls, for which there are microarray probes with perfect sequence complementarity (Figure S1). The sensitivity of deep sequencing is limited by the number of reads generated per sample, or read depth. In this study, we detected virus sequences down to two parts per million. Nearly every virus that was detected on the microarray was also detected by deep sequencing; additionally, in numerous samples (n = 44), sequencing revealed viruses not detected by the Virochip (Table 1). There were two instances where Virochip analysis identified a virus (TTV) that was not detected by deep sequencing (Table 1). Deep sequencing, therefore, is a superior method for novel virus discovery, because it is more sensitive and provides more conclusive genotypic information than the Virochip. Nevertheless, the Virochip is a relatively fast and inexpensive method that is best applied to samples with expected virus copy numbers present at levels greater than 1 in 105 host sequences.
We were unable to detect a virus in two thirds of the 123 dengue-like illness samples. These results could reflect true negative status, which would result from a non-viral infection, illness due to non-infectious agent, or complete immunologic clearance. Alternatively, the negative results could reflect failures in our diagnostic approaches due to imperfect sensitivity, unsatisfactory sample preparation, improper sample type, or failure to recognize highly divergent viral sequences. The presence of sequences that lack even remote similarities to known species also highlights the need for further development of de novo assembly methods for metagenomic data. Assembled data, increased depth, and enhanced sequenced comparison methods should enable more sensitive detection of divergent viruses in metagenomic samples.
Determining the etiology of human diseases with symptoms that overlap with dengue-like illness is important for understanding the full spectrum of emerging or previously uncharacterized pathogens in tropical populations. In this study, 10% of acute serum samples negative for dengue virus from cases of pediatric dengue-like illness were positive for HHV-6. Primary HHV-6 infection causes undifferentiated febrile illness and exanthem subitum (roseola infantum or sixth disease), an acute illness with high fever and rash that typically resolves in three to seven days. Exanthem subitum is a common disease of infants worldwide, and HHV-6 infection most frequently occurs between 6 and 12 months of age, with seropositivity estimates of >95% in adult populations in developed countries. The HHV-6 positive patients in this study were between 7–12 months old, and presented with fever and rash (Table S3). We detected multiple kilobases of HHV-6 sequence in each positive sample, with sequence deriving from multiple viral genomic regions (Figure 2).
After acute infection, HHV-6 can latently persist in the host quiescently, with no production of infectious virions or with low levels of viral replication. Latency is believed to endure in several cell types, including monocytes and bone marrow progenitor cells,, and may undergo chromosomal integration that can be vertically transmitted. The confounding effects of chromosomal integration make differentiating between active and latent HHV-6 infections difficult when detecting HHV-6 sequence in serum DNA,. A previous study detected integrated HHV-6 genomic sequence in ∼1% of healthy blood samples. Since detection of HHV-6 nucleic acid in serum alone does not prove active viral infection, we cannot definitively confirm that the HHV-6 sequences in these samples were not derived from the vertical transmission of chromosomally integrated virus. However, the clinical, epidemiological, and virus sequence data suggest HHV-6 may be the etiologic agent in these febrile illness cases.
Primary HHV-6 infection is a major cause (∼20%) of infant hospitalizations in the United States, a clinical burden likely shared throughout the tropical world given similar seroprevalence rates. The results of this study illustrate the importance of administering HHV-6 diagnostic tests to cases of suspected dengue-like illness in infants from dengue-endemic regions to differentiate between cases of exanthem subitum, a ubiquitous self-limiting childhood illness, and dengue fever, which carries a greater risk of severe clinical complications and death.
Similarly, the one sample positive for Parvovirus B19 sequence may be a case of acute infection with a commonly acquired childhood virus. Parvovirus B19 can manifest as erythema infectiosum (fifth disease), a condition associated with characteristic “slapped cheek” rash. Infection can also be subclinical or result in mild nonspecific symptoms. It is possible that Parvovirus B19 infection caused the symptoms in this case (Table S3), though as with HHV-6, the identification of viral sequences does not definitively demonstrate causality.
Epstein Barr Virus (HHV-4) sequences were found in the serum of one patient who presented with relatively severe symptoms, and died during hospitalization (Table S3). HHV-4 infection is a nearly universal occurrence in the first two decades of life,. Primary infection in adolescents or adults can manifest as infectious mononucleosis, and chronic infection is associated with various malignancies later in life. Primary infection during childhood, however, is usually asymptomatic or produces only mild symptoms. It is not clear that HHV-4 infection or HHV-4 alone caused the illness in this case.
In addition to the viruses for which a plausible disease association exists, many samples contained sequences from viruses with no well-established link to human disease. These included the two samples positive for GBV-C and those containing ASFV-like, TTV-like, and circovirus-like sequences.
The Circoviridae family is an extraordinarily diverse group of small, single-stranded circular DNA viruses that includes cycloviruses (genus Cyclovirus) and circoviruses (genus Circovirus), which are commonly detected in human stool and blood, and also in environmental samples–. Some circovirus species, such as beak and feather disease virus and porcine circovirus 2, have been associated with disease in bird and pig hosts, respectively, but the pathogenic potential of circoviruses in humans remains unconfirmed,. The circovirus-like sequences reported here were detected in nucleic acid libraries prepared from acute human serum and were most closely related to circovirus-like viruses (Figure 3), which were first reported in environmental samples and in bats,. We were unsuccessful in recovering a full genome sequence corresponding to any of the circovirus-like sequences, and it has not yet been possible to prove that these sequences were not an environmental artifact introduced during sample preparation. It is also possible that these sequences derive from other organisms, such as Giardia intestinalis or Entamoeba dispar, whose genomes encode proteins that share amino acid similarity with circovirus replicase proteins (Figure 3). Furthermore, it has yet to be established whether circoviruses are capable of replicating in humans. Pending additional screening and serologic studies, the detection of circovirus-like sequences from human serum should be interpreted with caution.
Metagenomic approaches provide an effective high-throughput method to detect uncharacterized virus diversity in a tropical setting from many samples simultaneously. The findings presented in this study further our knowledge of well-characterized and previously unknown viruses present in serum collected from pediatric dengue-like illness patients and advance our understanding of the application of metagenomic approaches to human pathogen detection. Deep sequencing analysis of clinical samples holds tremendous promise as a diagnostic tool by permitting the detection of many different viruses simultaneously, including those present at low-copy numbers and of divergent origin. Major remaining barriers to high-throughput sequencing strategies becoming standard diagnostic practice include prohibitive cost, lengthy sample preparation time, and computationally intensive data analysis requirements. These challenges are magnified in resource-limited settings, such as Nicaragua, but are gradually being addressed. Industry hardware and technical advancements have steadily decreased the per-base cost of deep sequencing, and the results presented here strengthen our expectations of multiplexed sample preparation and bioinformatic data filtering within the framework of current second-generation sequencing platforms. Long-term bi-directional partnerships with developing country collaborators facilitate easier access to techniques not currently available on-site, such as deep sequencing, and are also important in providing training opportunities for local scientists and developing relevant pathogen tests and diagnostic policies.
This study expands our understanding of the virus diversity in pediatric dengue-like illness in Nicaragua and the application of genomic detection techniques in a tropical setting, findings that are particularly valuable given the pressing need for improved global emerging pathogen surveillance.
The recent epidemic of Ebola virus in Africa as well as the emergence of a hitherto unknown virus known as Middle East respiratory syndrome coronavirus (MERS-CoV), Bas-Congo virus in central Africa or of severe fever with thrombocytopenia syndrome virus (SFTSV) in China have repeatedly shown the global impact of emerging infectious diseases (EIDs) on economics and public health. These EIDs, more than 60% of which are of zoonotic origin, are globally emerging and re-emerging with increased frequency. Surveillance and monitoring of viral pathogens circulating in humans and wildlife and the identification of EIDs at an early stage is challenging. Many potential emerging viruses of concern might already be infecting humans or wildlife but await their detection by disease surveillance. In remote and underdeveloped regions of the world, often no attention is paid towards possible infectious disease cases until a threshold of serious cases and deaths appears in a cluster and certain epidemic properties are reached. Some viruses might just be overlooked at population levels until they spread or re-emerge and become epidemic in another region or time. An effective strategy in virus surveillance would need to survey simultaneously a wide range of viral types in a large number of human and wildlife individuals in order to detect viruses before spreading. For example, the EcoHealth Alliance within the surveillance program PREDICT seeks to identify new EIDs before they emerge or re-emerge. Therefore, wildlife animals that are likely to carry viruses with zoonotic potential, e.g., bats, rodents, birds and primates, are sampled frequently. However, collecting swabs or blood from sufficient numbers of wildlife individuals and the subsequent identification of viruses is challenging. The solution for overcoming this challenge might be presented by the disease vector itself. Blood feeding arthropods feed on blood from a wide range of hosts including humans, mammals and birds. Therefore, they act as “syringes”, sampling numerous vertebrates and collecting the viral diversity over space, time and species. Xenosurveillance and vector-enabled metagenomics (VEM) are surveillance approaches that can exploit mosquitoes to capture the viral diversity of the animal, human or plant host the mosquito has fed on (Figure 1). Xenosurveillance, a term introduced by Brackney et al., refers to the identification of viral pathogens from total nucleic acids extracted from mosquito blood meals, either by next-generation sequencing (NGS) or conventional PCR assays. Recent developments in NGS and viral metagenomics, which is the shotgun sequencing of viral nucleic acids extracted from purified virus particles, offer great opportunities for the characterization of the complete viral diversity in an organism or a population. VEM, a technique used to sequence purified viral nucleic acids directly from insect vectors, has already been used to detect both animal and plant viruses circulating in vectors. This review summarizes findings from xenosurveillance efforts as well as VEM studies using mosquitoes, since both approaches combine sampling of multiple individuals of blood-feeding arthropods with the high-throughput properties of NGS.
In addition to the possible transmission of porcine bacteria and fungi, which may be eliminated using antibiotics and antimycotics, transmission of viruses through xenotransplantation may lead to disease in the recipient, i.e. zoonoses. For most porcine viruses there are neither antivirals nor vaccines available. However, it is still unclear whether porcine viruses can actually infect humans and cause zoonoses. In contrast to human pathogens that are well adapted to humans, porcine microorganisms are not. For only a few porcine viruses a zoonotic potential has been described, for example for HEV, genotype 3. HEV induces a chronic infection in immunosuppressed patients and severe disease in patients with a pre-existing liver failure (for review see [29, 30]). Of special interest is the porcine cytomegalovirus (PCMV) which may be indirectly pathogenic without infecting cells of the host. In preclinical trials of transplanting pig kidneys into cynomolgus monkeys and baboons, the presence of PCMV led to early transplant failure (for review see). Since there is still no evidence for PCMV infection of non-human primate as well as human cells, the organ failure was possibly due to cytokines produced in response to viral antigens.
Porcine lymphotropic herpesvirus (PLHV) 1, 2, and 3 are common porcine herpesviruses, but their prevalence and importance for xenotransplantation is not well understood [32, 33]. PLHV may be transmitted by pre-partum cross-placental vertical transfer or post-partum horizontal transmission, although the former is relatively rare. Between 26% and 88% of animals in different herds in Germany, Ireland, France, Spain and the United States are infected with one of the PLHV variants [34–37] and, unlike PCMV, early weaning cannot eradicate PHLV.
Porcine circoviruses (PCV), the smallest viruses replicating autonomously in mammalian cells, are also widely distributed (for review see). There are two types, porcine circovirus 1 (PCV1), which is apathogenic in pigs, and PCV2, which causes a severe multifactorial disease (PCV2 disease, PCVD, formerly post-weaning multisystemic wasting syndrome, PMWS). Although the presence of PCV2 is necessary for the disease, additional factors are also required and the severity of the disease depends on the status of the immune system and genetic predisposition. There is no serological evidence indicating transmission of PCV2 to humans and indeed, the case of a human rotavirus vaccine being contaminated with PCV demonstrated that PCV was not transmitted to the vaccinated humans [40, 41]. Infection experiments with human cell lines showed persistence of PCV1 in most cell lines without causing any visible changes, but cytopathogenic effects in PCV2-transfected cells. Most importantly, the infection appeared to be non-productive [42, 43].
It is interesting to note that porcine viruses with a known or suspected zoonotic potential such as the herpesviruses PCMV and PLHV were not detected when viromes were analysed in faeces and blood [7–10] whereas PCV (mainly PCV2) was commonly found [7–10]. Presumably, the frequency of the herpesviruses is so low that their DNA cannot be detected by next generation sequencing. Indeed, only low numbers of herpesvirus sequences were found in German sows [11, 12] and in swine intestinal mucus, the raw material for the manufacture of heparin. Similarly, next generation sequencing of human samples failed to identify low-level viruses that are revealed using viral particle enrichment or PCR amplification [44, 45]. The most effective approach was serological profiling, detecting virus-specific antibodies in human sera using a synthetic human virome. Testing 569 human donors across four continents with this method revealed antibodies to an average of 10 human viral species per person and 84 species in at least two individuals.
Whereas the zoonotic potential of many pig viruses is still unknown, some viruses are well characterised zoonotic viruses. One example is the hepatitis E virus (HEV) which can be easily transmitted by consumption of undercooked pork or by contact to pigs and which can induce disease and chronic infection in immunosuppressed individuals and in people with liver failure (for review see). A second example is the paramyxovirus Nipah virus, which was transmitted from pigs to humans mainly working in slaughterhouses in Malaysia and Singapore [47, 48]. The virus induced severe infections of the respiratory tract and encephalitis in humans, 48% of the infected died [47, 48]. The Menangle virus was first described in a piggery in Australia experiencing a high number of stillbirths and deformities during farrowing. Two workers at the piggery got infected and had a serious flu-like illness. Nipah virus and Menagle virus had their origin in fruit bats. Rabies is considered a rare disease in pigs, however an infection with the rabies virus is invariably fatal in all species including the human [49, 51]. Another rhabdoviruses, the vesicular stomatitis virus (VSV) can infect insects, cattle, horses and pigs and is zoonotic, inducing flu-like illness in infected humans. The Eastern equine encephalomyelitis virus (EEEV) and the Japanese encephalomyelitis virus (JEV) are two arboviruses infecting pigs. The transmission cycle of the JEV involves pigs as virus amplifiers and mosquitoes as vectors for transferring the virus between amplifying hosts and to dead-end hosts, i.e. humans, horses and cattle. In pigs, it can cause abortion and stillbirths. In an outbreak of EEEV infection in swine, 280 of 350 pigs died. Histopathologic changes in the brain, multifocal necrosis and inflammation in the myocardium were seen. Growth retardation was noticed in surviving pigs. Pigs can be protected from EEEV infection by a vaccine. Domestic swine in the Philippines have been discovered to host Reston ebolavirus (REBOV). Although REBOV is the only member of Filoviridae that has not been associated with disease in humans, its emergence in the human food chain is of concern.
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.
The genus Cosavirus consists of five species (Cosavirus A, B, and D to F), which have been associated with gastroenteritis in children (39). Six near-complete human cosavirus (HCoSV) genomes were identified: 1 from children less than 3 years old (HP49), 3 from those between 3 and <20 years old (HP6A and HP6B, HP57), and 2 from pools of individuals between 20 and <60 years old (HP44, HP24). Some of these pools had direct or indirect contact with bats (HP6, HP24, and HP44), while others had no contact with bats (HP49 and HP57). Phylogenetic analysis (Fig. 4C) showed that cosaviruses from HP6B, HP49, and HP57 formed a clade with two other strains from Australia and Nigeria (HCoSV/E1/AUS and HCoSV/NG385/NGA) in species HCoSV E. Meanwhile the strains in HP6A, HP24, and HP44 clustered with HCoSV in species A, D, and B, respectively. Therefore, it seems that humans in Cameroon host a diverse range of cosaviruses.
With the advent of next generation sequencing techniques, samples from a wide range of animal species have been screened to identify novel viruses and this approach has become the most important tool for early detection and characterization of possible emerging zoonotic agents [1–3]. It is important to monitor these emerging zoonotic agents as they can be responsible for minor or major epidemics worldwide. For example, zoonoses can range from the Middle East respiratory syndrome coronavirus (MERS-CoV), which recently drew a lot of attention worldwide, to the 2009 pandemic H1N1 influenza A virus or to the less publicized Hepatitis E virus. Not only are humans at risk, but also animals can be infected with viruses from other host species, resulting in disease, or further transmission to humans. The severe acute respiratory syndrome (SARS) coronavirus pandemic originated from wildlife, where bats where identified as the reservoir and civets as an intermediate host [7, 8]. A similar example comes from Hendra virus, which also originated from bats, but transmission to humans occurred with horses as an intermediate host, causing severe pneumonia in horses and humans. As such, zoonotic infections can have significant consequences for animal and public welfare. However, little is known about these pathogens before they emerge from unrecognized zoonotic sources and therefore a deeper understanding of the virome of wild animals will allow us to more rapidly identify the host of particular novel zoonotic viruses, and act appropriately to prevent further spread of such viruses.
Fourteen species of wild carnivores can be found in Portugal, often in relative close contact with humans. Only red fox, stone marten, badger, common genet and, more recently, the Egyptian mongoose, are known to have a generalized distribution throughout the country. From these animals we sampled two species, a badger (Meles meles) and an Egyptian mongoose (Herpestes ichneumon). In addition, we sampled a Eurasian otter (Lutra lutra) raised in a zoo, since otters are also widely distributed in Portugal. Currently little is known about the viral communities that populate the gut of these animals. Thus far, Bodewes and colleagues investigated the fecal virome of badgers and an otter from Spain, and found a fecal phlebovirus in an otter. A study by van den Brand and colleagues investigated the virome of badgers in the Netherlands, identifying two novel circularviruses. In Portugal, a study performed by Oliveira and colleagues screened otters for parvoviruses, adenoviruses and parainfluenza virus, however none of the samples had detectable levels of virus. Also in Portugal, another study screened for and identified parvoviruses in genets, badgers and mongooses. As these findings are probably only the tip of the iceberg, we were interested to further explore the viral communities of the gut in widely spread wild species in Portugal and a zoo specimen.
IgG antibodies to the novel bunyavirus were detected in 80 of 285 acute-phase serum samples from patients with FTLS (Table 5). Of 95 patients from whom paired acute- and convalescent-phase sera were available, 52 had seroconversions and 21 had greater than 4-fold increases in antibody titer to the virus. Six had less than a 4-fold increase in antibody titer to the virus, but all paired sera tested positive. Sixteen patients tested negative to the virus, suggesting that some non-FTLS patients with similar symptoms were included in this study, a situation that is not surprising given that FTLS is a newly emerging disease. The acute-phase sera of four patients from whom the virus was isolated tested negative for IgG antibody to the virus. All convalescent sera obtained 2 months later from the same four patients contained IgG antibody to the virus. None of the 130 sera from patients with respiratory diseases or healthy subjects had detectable antibody.
The genus Parechovirus is comprised of two species, Parechovirus A (human parechovirus [HPeV]) and Parechovirus B (Ljungan virus, isolated from bank voles) (33). HPeV is subdivided into 19 types (HPeV1 to -19). HPeV is associated with mild gastrointestinal or respiratory illness; however, severe disease conditions, such as meningitis/encephalitis, acute flaccid paralysis, and neonatal sepsis, may occur (34–36). Here, three (nearly) complete HPeVs were identified in pools HP2, HP46, and HP48 with sequence lengths of 7,142 bp, 7,202 bp, and 7,219 bp, respectively, collected from children less than 3 years old (age group A). In terms of bat contact status, they were in pools of those either in indirect contact with bats (HP2 and HP48) or without contact (HP46). They were all distantly related to each other, with HPeV-CMRHP46 and HPeV-CMRHP48 having the highest identity (76% and 86% nt and aa identity, respectively). Phylogenetically, HPeVs in HP46 and in HP48 fell into a clade of type 1 HPeVs (Fig. 4B). The HPeV in HP46 clustered together with HPeV1/Harris strain with 76% nt identity, while CMRHP48 clustered closely with Japanese and Norwegian strains A1086-99 and NO-3694 (84 to 90% nt identity). Furthermore, HPeV-CMRHP2 clustered distantly with type 16 HPeVs from China and Bangladesh with only 70 to 71% nt identity. Considering the 75% identity demarcation for HPeV types (37, 38), this strain potentially represents a novel type.
Previously, virus discovery in animals has focused on pathogenic infections or on animals that on the basis of relevance to (re-)emerging viruses are thought to represent a key risk host for emerging virus-associated disease in humans. With the recent advances in the metagenomics field, a substantial increase in studies looking at virus epidemiological baseline levels in different (wildlife) animals has been observed,,,–. Even though ferrets are a very important animal model for a number of human viral infections, not much is known about viruses that naturally occur in ferrets–. This study describes the viral communities in fecal material of ferrets (Mustela putorius furo).
Sequences closely related to known viral sequences were identified, with homology to ferret coronavirus, ferret hepatitis E virus, Aleutian mink disease virus, different avian viruses and murine astrovirus STL1,,. The avian viruses may be a reflection of the diet of these ferrets, which were fed chicken. It is of note that both the viral screening with random amplification and next-generation sequencing and a specific ferret hepatitis E virus taqman assay indicated that household ferrets that are kept as pets are significantly more likely to excrete ferret hepatitis E virus in their fecal material than farm animals in this study. Such a correlation was not observed for ferret coronavirus excretion for which prevalence in both cohorts seemed similar, but not as high as previously reported. Sporadic cases of human hepatitis E virus infections seem to be increasing and several observations suggest that these cases are caused by zoonotic spread of infection from wild or domestic animals–. Follow-up studies into seroprevalence and/or virus prevalence in household ferrets, their owners, and possibly other pets may provide indications for cross species transmission.
We report on the first identification of a kobuvirus, parechovirus, papillomavirus and anellovirus in ferrets. The kobu- and parechovirus belong to the family Picornaviridae that currently comprises 17 genera and many more different virus species. They infect a wide range of mammals and are well-known for their involvement in gastroenteritis. Our phylogenetic analysis revealed that the ferret kobuvirus clustered with bovine and ovine kobuvirus in species Aichivirus B. The close relationship between bovine, ovine, and ferret kobuviruses may indicate past cross species transmission events and subsequent evolution in the separate host species resulting in the distinct types seen today. As was observed for ferret hepatitis E virus but not ferret coronavirus, ferret kobuvirus is significantly more often detected in household ferrets than in farm ferrets by a specific ferret kobuvirus taqman assay, although random amplification and next-generation sequencing suggested the presence of kobuvirus in two additional samples from farm ferrets, which could reflect the presence of another divergent kobuvirus species. Also here follow-up studies into seroprevalence and/or virus prevalence in household ferrets, their owners, and possibly other pets should be performed. An overall kobuvirus prevalence of ∼15% was observed in this study. Two out of 3 animals (67%) with diarrhea and 4 animals out of 36 (11%) without diarrhea were positive by taqman for ferret kobuvirus. Because of the small sample size and the fact that other causes for diarrhea were not excluded, further in depth studies are needed to show that these viruses can cause diarrhea in ferrets.
The ferret parechovirus is the third parechovirus species identified to date. It is highly divergent from human parechovirus and Ljungan virus and may even constitute a new genus in the family Picornaviridae based on the observed sequence diversity. Interestingly, the P3 genome region of MpPeV1, encoding the nonstructural proteins NS3A–3D, is not more conserved than the P1 genome region, encoding the structural capsid proteins, when compared to the corresponding regions of human parechoviruses 1 and 2. This has been observed for Ljungan virus as well by us and others and requires more in-depth studies. Only 1 out of 39 ferrets was positive for MpPeV1, suggesting that the ferret parechovirus prevalence may be relatively low (<2.5%). In humans, parechovirus infections are most common in young children and the prevalence in adults may be low as well. It is of note that the parechovirus-positive animal did not show any signs of gastroenteritis. Low virus prevalence was observed for the identified ferret papillomavirus as well. Papillomaviruses are, however, not typically detected in fecal material or associated with gastroenteritis like parechoviruses. Although recently a full-length papillomavirus genome was characterized from fecal material of a deer mouse as well. Not much knowledge exists on papillomatosis in ferrets as a disease, or the virus(es) causing them. The impact of papillomavirus infections on ferrets in general and the specific role of MpPV1 thus has to be further addressed.
Like many other mammals, it seems that ferrets also harbor anelloviruses. The ferret anellovirus MpfTTV1 is most closely related to the recently identified anellovirus from pine marten. Based on phylogenetic analysis, we propose that MpfTTV1 should be placed in the proposed new anellovirus genus, Xitorquevirus, in analogy to the classification of torque teno viruses in nine genera named Alpha-, Beta-, Gamma-, Delta-, Epsilon-, Eta-, Iota-, Theta-, and Zetatorquevirus, and the proposed five genera Kappa-, Lambda-, Mu-, Nu-, and Xitorquevirus
,,. A closely related virus to MpfTTV1 seemed to be present in one other ferret (Table 1) besides ferret 21, resulting in a virus prevalence of ∼5% in this study. Anelloviruses do not generally seem to be as abundantly present in fecal material as in serum, as evidenced by fecal virome studies in different host species,,,,,,,.
Ferrets are carnivores that are significantly affected by a number of human pathogens and hence are widely used as a small animal model for viral infections–. In addition, they are capable of spreading viral infections to humans, among which influenza A virus and on rare occasions rabies virus–. The characterization of the fecal virome of ferrets provides epidemiological baseline information about pathogens which allows the swift identification of possible sources of future zoonotic infections and their subsequent control. Especially the seemingly interesting correlation between certain viral infections in ferrets and their presence in human households as pets needs to be further addressed. In addition, this study shows that ferrets can be infected with a wide range of different viruses, some of which may influence the outcome in experimental virus infection studies, thus delivering a strong argument in favor of development of specific pathogen-free ferret colonies.
Since 2007, there has been an increase in reported cases of FTLS in Xinyang City, Henan Province. These patients were tentatively diagnosed as having A. phagocytophilum infection. However, only a few (8.4%, 24/285) such patients had evidence for A. phagocytophilum infection, and none of the 285 patients tested positive for the many other pathogens capable of causing similar clinical and laboratory manifestations that were also investigated. These findings suggested novel infectious agents, including viruses.
Traditionally, virus culture is very important for identifying an unknown viral infection. Before performing the Illumina sequencing strategy, we attempted viral and rickettsial culture with DH82 and BHK cell lines, but the lack of an obvious CPE led us to initially abandon this approach. Here, mass sequence data obtained by Illumina sequencing revealed four virus families that appeared only in FTLS patient sera. Among these four virus families, viruses from the Parvoviridae and Bunyaviridae families reportedly can cause signs of FTLS and be transmitted by arthropods. However, only one sample from a pool of ten samples tested positive for bocavirus by PCR, suggesting that bocavirus from the Parvoviridae is not likely involved in FTLS. For viruses in the Bunyaviridae family, the incidence of infection is closely linked to vector activity. For example, tick-borne viruses are more common in the late spring and late summer when tick activity peaks. Human infections with certain Bunyaviridae, such as Crimean-Congo hemorrhagic fever virus, are associated with high levels of morbidity and mortality. Considering the tick-bite history of many FTLS patients, we focused on Bunyaviridae family viruses.
The entire Bunyaviridae family contains more than 300 members arranged in four genera of arthropod-borne viruses (Orthobunyavirus, Nairovirus, Phlebovirus and Tospovirus) and one genus (Hantavirus) of rodent-borne viruses,. The Phlebovirus genus currently comprises 68 antigenically distinct serotypes, only a few of which have been studied. The 68 known serotypes are divided into two groups: the Phlebotomus fever group (the sandfly group, transmitted by Phlebotominae sandflies) comprises 55 members, and the Uukuniemi group (transmitted by ticks) comprises the remaining 13 members. Of these 68 serotypes, eight are linked to disease in humans, including the Alenquer, Candiru, Chagres, Naples, Punta Toro, Rift Valley fever, Sicilian, and Toscana viruses. Phleboviruses have tripartite genomes consisting of a large (L), medium (M), and small (S) RNA segment.
In screening for unknown viruses, species hits alone likely carry little weight. Thus, we used all sequences in the family Bunyaviridae for our analysis. A 168-bp fragment of the polymerase gene with the lowest E-value and high sequence identity was used as the sequence of the unknown virus. This virus sequence was detected in all 10 pooled samples, indicating that the virus is involved in FTLS. After detecting a possible novel bunyavirus through high-throughput Illumina sequencing, we inoculated Vero cell lines, which are known to be sensitive to phleboviruses, with sera from six positive patients and were subsequently able to detect the virus by RT-PCR,. Although the CPE was modest, RT-PCR confirmed the infection. Genome sequencing was performed and a phylogenetic analysis of the genome sequence showed that this virus clustered into the Phlebovirus branch, but was divergent from other known phleboviruses. These results confirm the novelty of this virus within the Phlebovirus genus of the family Bunyaviridae
. Furthermore, virus size and propagation in cells were similar to that of the bunyaviruses.
PCR and serological tests were performed to further test the causal link between the new virus and FTLS. Although we have not completely fulfilled Koch's postulates, evidence implicating this new bunyavirus in the outbreak of the disease among patients with FTLS is compelling.
In view of the fact that the disease is caused by a novel bunyavirus, and taking into account that the disease was first discovered in Henan (HN), we propose the name "Henan Fever" for the FTLS disease cause by the novel virus (proposed name “Henan Fever Virus” [HNF virus]). Since the submission of this manuscript, a bunyavirus was identified as the cause of FTLS in Chinese patients from other regions of China, and the authors have named this virus “SFTSV” to indicate that it is the cause of severe fever with thrombocytopenia syndrome. After release of the GenBank sequences referred to in the Yu paper, we compared the sequences of SFTSV with those of FTLSV and found that they were nearly identical (>99% identity). As we first identified the syndrome in 2007 and described the presence of the virus in patients between 2007 and 2010, we suggest that the name “HNF virus” should take precedence. The most distinctive feature of the current work includes the use of an unbiased metagenomic approach for viral pathogen discovery that facilitated the rapid creation and implementation of standard culture, serological, and molecular diagnostic approaches. However, there are other differences between the results described here and those reported by Yu et al; notably, we observed slight, but distinctive, CPE in Vero cells. The reason for the failure to observe CPE in Vero cells infected with the “SFTSV” bunyavirus, whose genome is nearly identical to that of bunyavirus isolated from our FTLS patients, is unclear. Perhaps this reflects the fact that the ensuing CPE is not dramatic. Alternately, this could indicate the existence of distinct viral strains that vary in pathogenicity, virulence, and possibly even disease manifestations. This is an area of active study in our laboratories.
The discovery of this new virus will assist in the rapid diagnosis of this disease and help to distinguish it from other diseases caused by pathogens such as A. phagocytophilum, E. chaffeensis, Crimean-Congo hemorrhagic fever virus, Hantavirus, dengue virus, Japanese encephalitis virus, and Chikungunya virus. Furthermore, the availability of the new virus will facilitate the future development of new therapeutic interventions, such as vaccines and drugs.
Canine enteritis can be caused by a number of viral, bacterial or parasitic agents. The most common viral entero-pathogens are canine parvovirus (CPV) and coronavirus (CCoV),, although other agents, such as canine adenovirus (CAdV) type 1, canine distemper virus (CDV), rotaviruses, reoviruses, and caliciviruses, have been associated with enteric disease in dogs. In recent years, novel viruses have been discovered from dogs with enteritis, namely noroviruses, sapoviruses, astroviruses, and kobuviruses,.
More recently, a dog circovirus (DogCV) was detected in dogs with vasculitis and/or hemorrhagic diarrhoea in the US (13). Circoviruses (family Circoviridae, genus Circovirus) are non-enveloped, spherical viruses with a small monomeric single-strand circular DNA genome of about 2 kb in length. According to the most recent release of the Universal Virus Database of the International Committee on Taxonomy of Viruses, the genus Circovirus consists of eleven recognized species, including Porcine circovirus 1 (PCV-1), Porcine circovirus 2 (PCV-2), Canary circovirus (CaCV), Beak and feather disease virus (BFDV), and other viruses of domestic and wild birds (http://ictvdb.bio-mirror.cn/Ictv/fs_circo.htm). Porcine and avian circovirus infections are characterized by clinical courses that may vary from asymptomatic infections to lethal disease.
Two independent studies have shown that, similar to other animal circoviruses, DogCV possesses an ambisense genomic organization with 2 major inversely arranged ORFs encoding for the replicase and capsid proteins, respectively,. The canine virus, firstly detected in serum samples, was later recognized as causative agent of necrotizing vasculitis and granulomatous lymphadenitis.
The aim of this paper is to report the detection and molecular characterisation of DogCV in dogs with acute gastroenteritis in Italy. The full-length genome of the Italian prototype strain was determined and analyzed in comparison with American strains and other circoviruses.
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 recent years, a number of novel canine viruses have been discovered. DogCV, first detected in dogs' sera in 2012, was later associated to canine vasculitis and hemorrhage. However, analysis of archival samples has revealed that this novel virus has been circulating in dogs for at least 5 years before being discovered. The data gathered in this study extend the geographical distribution of DogCV to the European continent. In addition, our findings suggest a possible association between DogCV and canine enteritis. Unfortunately, the carcass of the CV-infected dog had been stored frozen for some months before being delivered to our laboratory. Long-term storage at low temperatures hindered the execution of histopathology, and valuable information on tissue localization and alterations of DogCV could not be gathered.
The full-length genome of the Italian prototype strain Bari/411–13 was determined, revealing close genetic relatedness to the American viruses. Virus isolation attempts using different canine cell lines were unsuccessful although the CT values obtained in real-time PCR amplification were suggestive of high viral titers in the inocula. PCVs have been reported to grow better in the presence of glucosamine, which may help the virus enter the cell nuclei, but this procedure was found not to be helpful for DogCV isolation.
In swine, while PCV-1 is almost completely apathogenic, PCV-2 has been proven to cause a systemic disease known as postweaning multisystemic wasting syndrome (PMWS), often in association with other porcine pathogens. However, since PCV-2 is ubiquitous and the disease observed in the field was not successfully reproduced under experimental conditions, several years passed before the scientific community reached a full agreement about its role as causative agent of PMWS.
A number of novel circoviruses have been detected in recent years, including human and animal viruses that have been proposed to form the new genus Cyclovirus of the family Circoviridae
. However, very little is currently known about the ability of these novel circoviruses to cause disease in their natural hosts. The present report, if corroborated by further reports, could represent a useful contribution to clarify better the pathogenic potential of DogCV, even if further studies are needed to assess the pathogenic and epidemiological features of this novel canine agent.
Infectious viral diseases, both emerging and re-emerging, pose a continuous health threat and disease burden to humans. Many important human pathogens are zoonotic or originated as zoonoses before adapting to humans–. This is exemplified by recently emerged diseases in which mortality ranged from a few hundred people due to infection with H5N1 avian influenza A virus to millions of HIV-infected people from acquired immunodeficiency syndrome–. Severe acute respiratory syndrome (SARS) coronavirus and the pandemic influenza A/H1N1(2009) virus in humans were linked to transmission from animal to human hosts as well and have highlighted this problem–. An ongoing systematic global effort to monitor for emerging and re-emerging pathogens in animals, especially those in key reservoir species that have previously shown to represent an imminent health threat to humans, is crucial in countering the potential public health threat caused by these viruses.
Relatively few studies have been conducted on diseases of non-domestic carnivores, especially regarding diseases of small carnivores (e.g. mustelids). Ferrets (Mustela putorius furo) can carry bacteria and parasites such as Campylobacter, Giardia, and Cryptosporidium in their intestinal tract and potentially spread them to people,. In addition, they can transmit influenza A virus to humans and possibly on rare occasions rabies virus as well–. Because of their susceptibility to several human respiratory viruses, including human and avian influenza viruses, SARS coronavirus, nipah virus, and morbilliviruses–, ferrets have been used as small animal model for these viruses. To further characterize this important animal model and to obtain epidemiological baseline information about pathogens in ferrets, the fecal viral flora of ferrets was studied using a metagenomics approach. Both known and new viruses were identified.
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).