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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.
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.
Approximately two thirds of emerging infectious diseases (EIDs) that affect humans originate from bats, rodents, birds, and other wildlife [1–3]. In many of these reservoir host species, emerging viruses appear to be well adapted, with little or no evidence of clinical disease. However, when these viruses spill over into humans, the effects can sometimes be devastating [4–6]. Previously, our limited knowledge of the viral population and ecological diversity harbored by wildlife have complicated the study of EIDs. Thus, comprehensive understanding of the viral community present in wildlife, as well as the prevalence, genetic diversity, and geographical distribution of these viruses, could be valuable for prevention and control of wildlife-origin EIDs.
The order Rodentia is the largest mammalian order, with 33 families and 2,277 species (~ 43% of all mammal species). They live in close contact with humans and their domestic animals and act as a bond between humans, domestic animals, arthropod vectors (ticks, mites, fleas), and other wildlife [8–10]. This interface with humans has led to the rodent origin of important zoonotic viruses including members of the family Arenaviridae, Hantaviridae, Reoviridae, Togaviridae, Picornaviridae, and Flaviviridae [11–18]. Many of these viruses cause severe disease in humans (e.g., Lassa virus; tick-borne encephalitis virus, TBEV; lymphocytic choriomeningitis virus, LCMV; Sin Nombre virus; Hantaan virus, HTNV; Seoul virus, SEOV; and Puumala virus); have only recently been discovered (e.g., Whitewater Arroyo virus and Lujo virus); or appear to have a wider geographical range than originally thought (e.g., Junin virus, Guanarito virus, Machupo virus, and Sabia virus), suggesting that further viral discovery studies in wild rodent populations may be valuable for public health [8, 11–13, 15, 19–25]. Recent reports of rodent viruses have enabled new hypotheses regarding the evolution of hepaciviruses and the origin of coronaviruses (CoVs) and picornaviruses (PicoVs) such as hepatitis A virus [26–29].
China is a megadiversity country and harbors ~ 200 rodent species from 12 families. To develop baseline data on the origin of existing viral EIDs and identify other potential zoonotic viral reservoir hosts, we have conducted a series of viral surveys from rodents, bats, and other small animals and have simultaneously constructed online viral databases of these animals (DBatVir and DRodVir, http://www.mgc.ac.cn/) since 2010 [31–34]. In the current study, 3,055 small mammal individuals of 55 species from the orders Rodentia, Lagomorpha, and Soricomorpha across China were sampled by pharyngeal and anal swabbing. Virome analysis was then conducted to outline the viral spectrum within these samples. On the basis of virome data, we describe the community, genetics, evolution, and ecological distribution characteristics of viruses and determined whether these features change with their host species and locations. The identification of novel mammal viruses provides new clues in the search for the origin or evolution pattern of human or animal pathogens such as hantaviruses (HVs), arenavirus (AreVs), CoVs, and arteriviruses (ArteVs).
The porcine circovirus (PCV) belongs to the family Circoviridae and contains a single-stranded circular DNA genome. There are three types of PCV: porcine circovirus type 1 (PCV1), porcine circovirus type 2 (PCV2) and porcine circovirus 3 (PCV3). During the past few decades, PCV2 has been widely studied and is considered to be the main pathogen responsible for porcine circovirus diseases and porcine circovirus-associated diseases (PCVD/PCVAD), which are characterized as clinical or subclinical PCV2 infections among pigs. The most representative symptoms of the diseases include porcine dermatitis and nephropathy syndrome (PDNS), which mainly occurs during the growing or finishing stage of pigs; postweaning multisystemic wasting syndrome (PMWS), which affects nursery and growing pigs; and porcine respiratory disease complex (PRDC), which usually occurs in pigs 14–20 weeks of age.
To date, the exact mechanisms of PCVD/PCVAD are currently unknown. However, many studies have reported co-infection with other swine pathogens, such as porcine reproductive and respiratory syndrome virus, porcine parvovirus, swine influenza virus, Mycoplasma hyopneumoniae, and Salmonella spp., are important cofactors that may enhance PCV2 infection and the severity of PCVD/PDVAD. Furthermore, vaccination failure, stress or crowding together with PCV2-infected animals also cause PCVD/PCVAD. As co-infections with viruses are frequently detected in domestic pigs and wild boars, we discuss co-infections of pigs with PCV2 and other swine viruses in this review. Furthermore, co-infections of different PCV2 strains, which cause recombination and genomic shifts in recent years, are also reviewed.
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.
Diarrhea is the second most common cause of death worldwide and accounts for about 8 to 9% of the 5.9 million yearly deaths in children under the age of 5 (1, 2). Most of these deaths occur in Southeast Asia and sub-Saharan Africa (3, 4). The chances of infection with enteric viruses are higher in developing countries than developed countries, probably due to suboptimal sanitation and hygienic conditions and low quality of drinking water, especially in rural areas (5). In Cameroon, a limited number of studies have investigated the prevalence of enteric pathogens as the cause of gastroenteritis in humans. These studies mainly focused on the epidemiology of a limited number of pathogens such as rotavirus, norovirus, and enteroviruses, revealing significant differences in the prevalence of these viruses in different settings and time periods (4, 6, 7). In parts of Cameroon, a high prevalence of several enteric viruses such as enterovirus, norovirus, rotavirus, and adenovirus was found in children and adults (8). Generally in Africa, many episodes of gastroenteritis remain unexplained as no etiological agent is determined (9, 10). A proportion of the unexplained gastroenteritis cases are likely due to other known viruses, for which no tests were performed. However, a part of these gastroenteritis cases could also be caused by novel viral agents.
Transmission of these enteric viruses is predominantly fecal-oral, and humans are constantly exposed to these viruses through various routes (11). One of these routes is zoonosis from reservoirs in wild or domestic animals, either by insect vectors or by exposure to animal droppings or tissues. One rich but, until recently, underappreciated reservoir of emergent viruses is bats. Of the ∼5,500 known terrestrial species of mammals, about 20% are bats (12). Several viruses pathogenic to humans are believed to have originated in bats over the last several years, including severe acute respiratory syndrome (SARS)- and Middle East respiratory syndrome (MERS)-related coronaviruses, as well as filoviruses, such as Ebola and Marburg viruses, or henipaviruses, such as Nipah and Hendra viruses (13–18).
In the Southwest region of Cameroon, bats are hunted and eaten. Such close interactions provide ample opportunity for zoonotic events to occur (19).
Previously, we identified a plethora of known and novel eukaryotic viruses in Cameroonian fruit bats using a viral metagenomics approach, including viruses known to cause gastroenteritis in humans (sapovirus, sapelovirus, and rotaviruses A and H) and those not yet associated with gastroenteritis (bastrovirus and picobirna-like viruses) (20–23). In the current study, we metagenomically screened 221 human fecal samples collected in the same region (where bats are hunted and eaten), to assess (i) if any viruses of animal origin could be identified and (ii) which known human gastrointestinal viruses were present. These fecal samples were collected from children less than a year old to adults of more than 60 years who had gastroenteritis and/or were in contact with bats. Additionally, since the gut virome typically contains both eukaryotic and prokaryotic viruses (phages), of which the latter usually represents the largest fraction of the gut virome, we also analyzed the phageome of these samples.
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.
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.
The median age of patients was 57.2 years (range, 23–88) and the male-to-female ratio was 1 to 2.27; 219 patients (92.02%) were farmers and 19 (7.98%) were workers or students. Among patients, 52 (21.85%) reported a tick bite within 2 weeks (5–14 days) before the onset of clinical manifestations; the remaining patients did not recall receiving a tick bite.
The main clinical features in confirmed patients included sudden onset of fever (>37.5°C −40°C) lasting up to 10 days, fatigue, anorexia, headache, myalgia, arthralgia, dizziness, enlarged lymph nodes, muscle aches, vomiting and diarrhea, upper abdominal pain, and relative bradycardia (Table 1). A small number of cases suffered more severe complications, including hypotension, mental status alterations, ecchymosis, gastrointestinal hemorrhage, pulmonary hemorrhage, respiratory failure, disseminated intravascular coagulation, multiple organ failure, and/or death. Most patients had a good outcome, but elderly patients and those with underlying diseases, neurological manifestations, coagulopathy, or hyponatremia tended to have a poorer outcome.
Laboratory tests showed that confirmed patients characteristically developed thrombocytopenia, leukopenia, proteinuria, and elevated serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels (Table 2). Biochemical tests revealed generally higher levels of lactate dehydrogenase, creatine kinase, AST and ALT enzymes, especially AST.
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.
The evolution of emerging diseases is associated with factors embedded in the concept “host-agent-environment triangle” (1). To infect the host and cause disease, the pathogen needs to evade host defenses, which may occur through single point mutations, genome rearrangements, recombination and/or translocation (2). Genetic uniformity generated through genetic selection of the host (3) and the fact that demographic changes, intensification of farming, and international commerce have occurred markedly over the last decades, must be also considered as essential factors for the development of emerging diseases (4–6).
As well as in humans, emerging diseases drastically affect animal populations, especially food-producing animals. Livestock production in large communities (i.e., pig farms or poultry flocks) represents an excellent environment to facilitate the transmission and maintenance of huge viral populations, contributing to the pathogen evolution (through mutation, recombination and reassortment, followed by natural selection) (7–9). The intensification of livestock during the last four decades has probably been one of the main factors that contributed to the emergence of new pathogens and/or pathogen variants, leading to changes in the epidemiology and presentation of diseases (10).
The number of viral infectious diseases in swine has significantly increased in the last 30 years. Several important worldwide distributed viruses have been reported in this period, including Porcine reproductive and respiratory syndrome virus (PRRSV, family Arteriviridae), Porcine circovirus 2 (PCV-2, family Circoviridae) and Porcine epidemic diarrhea virus (PEDV, family Coronaviridae). In addition to those worldwide widespread viruses, an important number of novel swine pathogens causing different types of diseases has been described (11, 12). Although their economic impact might be variable, they are considered significant infection agents and their monitoring is nowadays performed in some parts of the world. Among others, relevant examples are Porcine deltacoronavirus (associated with diarrhea) (12), Senecavirus A (causing a vesicular disease and increased pre-weaning mortality) (11), Porcine sapelovirus (found in cases of polioencephalomyelitis) (13), Porcine orthoreovirus (assumed to cause diarrhea) (14), Atypical porcine pestivirus (cause of congenital tremors type II) (15) and HKU2-related coronavirus of bat origin (associated with a fatal swine acute diarrhea syndrome) (16).
Besides overt emerging diseases of swine, many other novel infectious agents have been detected in both healthy and diseased animals, and their importance is under discussion. This group of agents is mainly represented by Torque teno sus viruses, Porcine bocavirus, Porcine torovirus and Porcine kobuvirus, which are thought to cause subclinical infections with no defined impact on production (13, 17, 18). An exception may be represented by Hepatitis E virus (HEV); although it seems fairly innocuous for pigs, it is considered an important zoonotic agent (19, 20). Recently, a novel member of the Circoviridae family named Porcine circovirus 3 (PCV- 3), with unknown effects on pigs, has been discovered (21, 22).
Porcine circovirus 3 (PCV-3) was first described in 2015 in North Carolina (USA) in a farm that experienced increased mortality and a decrease in the conception rate (21). Sows presented clinical signs compatible with porcine dermatitis and nephropathy syndrome (PDNS) and reproductive failure. In order to identify the etiological pathogen, aborted fetuses and organs from the affected sows were collected for further analyses. Whilst histological results were consistent with PCV-2-systemic disease, both immunohistochemistry (IHC) and quantitative PCR (qPCR) methods to detect PCV-2 yielded negative results. Samples were also negative for PRRSV and Influenza A virus. Homogenized tissues from sows with PDNS-like lesions and three fetuses were tested through metagenomic analysis, revealing the presence of an uncharacterized virus (21). Further analyses using rolling circle amplification (RCA) followed by Sanger sequencing showed a circular genome of 2,000 nucleotides. Palinski et al. (21) also performed a brief retrospective study through qPCR on serum samples from animals clinically affected by PDNS-like lesions (but negative for PCV-2 by IHC) and pigs with porcine respiratory diseases. Results revealed PCV-3 qPCR positivity in 93.75 and 12.5% of the analyzed samples, respectively (21).
Interestingly, almost concomitantly, another research group from the USA reported a clinical picture pathologically characterized by multi-systemic and cardiac inflammation of unknown etiology in three pigs of different ages ranging between 3 and 9 week-old (22). Several tissues from these animals were tested by next-generation sequencing (NGS) methods and PCV-3 genome was found. Beyond NGS, in situ hybridization was performed in one out of these three pigs, confirming PCV-3 mRNA in the myocardium (cytoplasm of myocardiocytes and inflammatory cells mainly, although to a very low frequency).
Based on these two initial works, the name PCV-3 was proposed as the third species of circoviruses affecting pigs, since pairwise analysis demonstrated significant divergence with the existing PCVs. The novel sequences showed < 70% of identity in the predicted whole genome and capsid protein amino acid (aa) sequence compared to the other members of the Circovirus genus (22). Taking into account the economic importance and the well-known effects of PCV-2 on the swine industry, a new member of the same family like PCV-3 should not be neglected. Studies on epidemiology, pathogenesis, immunity and diagnosis are guaranteed in the next few years, but the scientific community is still in its very beginning on the knowledge of this new infectious agent. Therefore, the objective of the present review is to update the current knowledge and forecast future trends on PCV-3.
Canine morbillivirus (canine distemper virus, CDV) causes canine distemper (CD) in a wide range of mammalian hosts, and may produce systemic, respiratory, cutaneous, bone, and/or neurological manifestations in these animals1,2. CDV produces immunosuppression3 in susceptible hosts by targeting cells that express the signalling activation molecule (SLAM)4, which frequently results in opportunistic infectious diseases caused by agents such as Bordetella bronchiseptica5,6, Candida sp.7, Clostridium piliforme8, Toxoplasma gondii9–11, Dirofilaria immitis11, Mycoplasma cynos12, and Talaromyces marneffei13. Although the occurrence of CD is significantly reduced in domestic dog populations in developed countries due to the use of vaccination14, the disease is endemic and a major cause of canine mortality in urban populations of Brazil15,16, where an estimated 147.5–160.3 million USD is spent annually due to the therapy of the systemic effects of CDV15.
CDV has been diagnosed concomitantly with traditional viral infectious disease agents such as canine parvovirus-2 (CPV-2)17,18, canid alphaherpesvirus-118,19, canine adenovirus-1 and -2 (CAdV-1)20, and (CAdV-2)18,21 in dogs. Moreover, recently CDV has been identified in dogs simultaneously with emerging viral infectious agents including Canine kobuvirus22, Canine pneumovirus23, and Canine respiratory coronavirus6,23. Additionally, studies have detected canine infectious disease agents due to the amplification of nucleic acids in symptomatic6,23–25 and asymptomatic19 dogs by molecular assays. Alternatively studies have combined the pattern of organ disease observed by histopathology with electron microscopy20, immunohistochemistry (IHC)8,12,21,22,25,26 and/or the molecular identification8,10,12,18,22,27 of infectious disease agents of dogs.
Previous studies by our group8,10,18 and others12,21,26,27 have demonstrated the concomitant participation of several infectious disease agents in the development of diseases in dogs, principally puppies. It is proposed that puppies are probably more frequently coinfected by several infections disease agents than has been previously reported, particularly if there is the simultaneous involvement of CDV, and coinfections may result in the death of the affected dog due to multiple organ failure10. The objectives of this retrospective study were to evaluate the frequency of concomitant traditional infectious disease agents in the development of infectious diseases in puppies, correlate the presence of these pathogens with histopathologic patterns, and review specific aspects of the pathogenesis involving these infectious disease agents.
The virome is the community of viruses found in a particular ecosystem. Viromes characterized from animals and human are comprised of both prokaryotic and eukaryotic viruses. Commensal bacteriophages, which make up the major fraction of the fecal virome, can modulate the microbial community in the host body and influence host immunity. Although typically a smaller fraction of the enteric virome, mammalian viruses may cause diseases such as diarrhea resulting in malnutrition and dehydration. Deep sequencing of wild animal fecal viromes also unveiled many eukaryotic viruses whose pathogenicity, if any, remain mostly unknown. In the past, emergences of human infectious diseases have been initiated by zoonotic viruses originating from bats, rodents, and non-human primates. Ebola virus likely from bats, human immunodeficiency virus (HIV) from chimpanzees, and the Middle East respiratory syndrome coronavirus (MERS-CoV) from camels, have caused very large economic and public health disruptions. Therefore, it is important to identify the viruses within animals with the potential to spill over into human and result in pathogenic infections. Such zoonoses may take different routes including fecal-oral transmission. Outbreaks of zoonotic enteric viruses belonging to the families of Picornaviridae, Adenoviridae, Caliciviridae, and Reoviridae cause important enteric diseases in humans. Moreover, alteration of enteric virome in humans also affect bacterial microbiome stability and influence diseases such as inflammatory bowel disease and ulcerative colitis. Studies of intestinal and fecal bacterial communities have received much attention relative to that of the gut virome.
Cynomolgus macaque, a non-human primate species widely distributing across Southeast Asian countries have long been used for biological research including on influenza virus, Ebola virus, and simian/human immunodeficiency virus (SIV/HIV). The National Primate Research Center of Thailand–Chulalongkorn University (NPRCT-CU), maintains a colony of cynomolgus macaques captured from disturbed natural habitats. Although well-established biosecurity protocols are used to screen infectious viruses such as herpes B virus, simian retrovirus (SRV), simian immunodeficiency virus (SIV), simian-T-lymphotropic viruses (STLV) and foamy virus that might cause a sporadic outbreaks, the transmission of other viruses from wild-originating macaques remains possible. In addition, captivity may also influence gut microbiome and virome. A recent study illustrated that replacing the gut microbiome of inbred laboratory mice with that of wild mice restored their immune responses to better mimic those of wild animals. Here, we characterized and compared the fecal virome of wild and captive macaques and identified novel macaque viruses.
Multisystemic inflammation and myocarditis were initially linked with the presence of PCV-3 (22). One single study described PCV-3 in weaned pigs that suffered from gastro-intestinal disorders (diarrhea), showing higher prevalence in pigs with clinical signs (17.14%, 6 out of 35) compared to those with non-diarrhea signs (2.86%; 1 out of 35) (87). In another report, animals with congenital tremors were analyzed and PCV-3 was the only pathogen found in the brain, with high amount of viral DNA (101).
A total of 221 human fecal samples (131 from Kumba and 90 from Lysoka) were collected from two hospitals in the Southwest region of Cameroon, for viral metagenomics screening. From these fecal samples, a total of 63 pools were constituted in categories based on age, bat contact status, and location (see Table S1 in the supplemental material). Illumina sequencing of all the 63 human pools generated in total approximately 708 million raw paired-end (PE) reads (between 4.3 and 53.4 million reads per pool). After trimming, 67% of the reads (471 million) were retained and 86% of these retained trimmed reads (405 million) were annotated using Diamond. Of these, 18% (74 million) could be attributed as viral.
There was no difference in the gender (females, 7; males, 8) of the puppies during this study. Pure breed dogs (73.3%; 11/15) were predominant (Table 1) relative to their mixed breed counterparts (26.7%; 4/15). However, when the head conformation was considered within the purebred dogs28,29, most (54.5%; 6/11) were mesocephalic (medium-headed), followed by the brachycephalic (short-headed) breeds of dogs (36.4%; 4/11), and only one (9.1%) dolichocephalic dog. Additionally, most (72.7%; 8/11) of these were representatives of toy breeds, with only three large breed dogs. Furthermore, most (n = 5) of the cases occurred in 2013, followed by 2014 (n = 3), 2015 (n = 3), and 2017 (n = 3), with only one in 2016.
The principal clinical manifestations described are resumed in Table 1. Bloody diarrhoea (n = 11) was the most frequently described clinical manifestation, followed by anorexia (n = 5), abdominal pain (n = 4), and convulsions (n = 3). One puppy died (#12) without presenting any reported clinical manifestation. The course of clinical manifestations was acute in all puppies, varied between 1–10 days, and resulted in the spontaneous death of all puppies. The immunization history of these puppies was not known.
All 285 patients with FTLS were from the Henan Province of China and were provisionally diagnosed as suspected HGA on the basis of similar clinical manifestations,. They represented four different epidemiologically linked sporadic cases and a few clusters of cases including 79 patients in 2007, seven patients in 2008, 47 patients in 2009, and 152 patients in 2010. The patients presented mainly between April and October, peaking in April-May during the tea-picking season in Henan. All patients resided in mountainous and hilly rural areas. In our study, 238 of 285 patients tested positive for novel bunyavirus infection by RT-PCR and/or IFA.
Pharyngeal and anal swabs were collected from 3,055 individual small mammals captured from July 2013 to July 2016 in 20 provinces across China (Fig. 1a and Additional file 1: Table S1). These comprised 50 rodent species of the families Muridae, Cricetidae, Sciuridae, Dipodidae, Chinchillidae, and Gliridae; two lagomorphs of the family Ochotonidae; and three soricomorphs of the family Soricidae that reside in urban, rural, and wild areas throughout China. The most common species sampled were Apodemus agrarius, Niviventer confucianus, Rattus norvegicus, Rattus tanezumi, Rattus losea, and Sorex araneus. Due to repeated sampling of some species in the same location, swabs were combined into 110 pools for analysis.
PCV2 is divided into five genotypes according to the Cap gene sequence: PCV2a, 2b, 2c, 2d, and 2e. Moreover, the PCV2b genotype is classified into three clusters, 1A to 1C, and the PCV2a genotype is subdivided into five clusters, 2A to 2F. Recently, a retrospective study of PCV2 infection between 1996 and 1999 in China revealed a novel genotype PCV2f which shared lower sequence identity with the other known genotypes. Since the discovery of PCV2 in the late 1990s, the virus has continued to evolve, and two major genotype shifts have been observed. The first genotype shift in PCV2 was from PCV2a to PCV2b in 2004/2005. Since 2012, the predominant PCV2b has been gradually replaced by the PCV2d genotype in North America, China, South Korea and Uruguay. Besides, PCV2F becomes the predominant genotype in the PCV2a cluster in China.
It has been reported that concurrent infections with different PCV2 genotypes have been detected in the same pig, resulting in inter- and intra-genotype recombination. One hundred and eighteen PCV2-positive DNA samples isolated from diseased pigs were analyzed using a modified differential polymerase chain reaction (PCR) assay, and the results indicated that the coexistence rates of PCV2 genotypes were 32.2% (38/118) in sick pigs. The sequencing results of 38 co-infected samples showed that the coexisting genotypes were PCV2a-PCV2b (12/38), PCV2a-PCV2d (15/38) and PCV2e-PCV2d (11/38). One group reported that the recombination rate of the PCV2 isolates was 27.7% (17/54) in the samples collected from 2006 to 2016 in China, and the recombination mainly occurred in the ORF1 gene of PCV2. Furthermore, co-infections with different PCV2 genotypes may cause more serious disease. In cells infected with replicating viruses, both PCV2a and PCV2b genotypes were equally present. Further studies have demonstrated that pigs with dually heterologous inoculation or naturally infected with multiple PCV2 genotypes or strains displayed more severe lesions. These results suggest that the coexistence of different strains of PCV2 might contribute to the development of more severe clinical symptoms in pigs and more recombination events between strains in the field. Therefore, more studies need to focus on analyzing the recombination trends of PCV2 strains, which may provide a better strategy for vaccine development and vaccination strategy.
The genus Gyrovirus, a diverse group of non-enveloped icosahedral viruses containing circular single-stranded DNA, infects a wide range of hosts. They also trigger several serious diseases in animals as causative agents. In particular, chicken anaemia virus (CAV), a member of family Anelloviridae genus Gyrovirus, is the etiological agent of chicken infectious anaemia. CAV infects several bone marrow-derived cells which results in severe anaemia and immunosuppression in young chickens. In terms of older birds, CAV can jeopardize the immune responses [3, 4]. Since its first reported in 1979, CAV infection has become epidemic among chicken populations on a global scale [5–7]. CAV also has dramatic financial impact in areas of intensive chicken farming. Vaccination is generally used to contain the spread of the virus. In a recent study, a novel human gyrovirus was isolated from a skin swab and designated as human Gyrovirus (HGyV). Since Circovirus shares partial homology to CAV, the identification of HGyV signals possible threats for human pathogenesis, further investigation is yet required.
The negative-sense CAV genome consists of 2,319 nucleotides and is replicated by a rolling-circle mechanism; but the packaging and egress of viral particles are poorly characterised [1, 10]. The CAV genome encodes multiple overlapping open reading frames (ORFs) that are translated into three main distinct polypeptides: CAV viral protein 1 (VP1, 52 kDa), viral protein 2 (VP2, 24 kDa) and viral protein 3 (VP3, 16 kDa). VP1 is the major structural protein while the VP2 is a replicase with dual-specificity phosphatase activity. VP3, also named apoptin, is also a non-structural protein that mainly implicats in the induction of apoptosis and viral cytotoxicity in host cells.
In 1996, CAV was first reported from young broilers in China. 42% of overall seroprevalence was shown in farms of five Chinese provinces in a domestic poultry survey. In addition, a high prevalence of 87% resulted in studies of the virus on live bird markets in Southeast China.
In the present study, our group investigated the epidemiology of CAV in sick or dead chickens in 12 provinces throughout China from 2014 to 2015. Totally, we obtained 96 positive results for CAV infection in 722 clinical samples, 24 out of 149 in 2014, and 72 out of 573 in 2015. We analysed the infection type of CAV in association with other pathogens including Marek’s disease virus (MDV), reticuloendotheliosis virus (REV), avian leukosis virus (ALV), avian gyrovirus 2 (AGV2), and avian reovirus (ARV). We found that coinfection was the main infection type of CAV. In addition, we analysed the characteristics of the new CAV sequenced strains together with those available in GenBank. The analysis revealed that all the sequences could be clustered into four major groups. Furthermore, we compared the key amino acids in VP1 that determined the virulence of CAV, providing new insights into the epidemiology of CAV.
Xenotransplantation is being developed to overcome the shortage of human tissues and organs needed to treat organ failure by allotransplantation. Pigs are the preferred species to be used as donor animals for a number of reasons including the size of the animals, their similar physiology, and the ease with which they can be genetically modified and cloned.
Although there are still some hurdles that have to be overcome, such as immunological rejection, physiological incompatibility and risk of transmission of porcine microorganisms, recent achievements in breeding genetically modified pigs, in the development of new immunosuppressive regimens and in approaches to safety suggest that xenotransplantation may soon be introduced in the clinic (for review see [1–3]).
In the past, only a few porcine viruses with known or suspected ability to infect human cells or to accelerate transplant rejection have been analysed within the context of xenotransplantation. Next generation sequencing or deep sequencing techniques and metagenomic analyses now give the opportunity to analyse the entire virome of pigs and its impact on xenotransplantation. These studies on the pig virome are, like investigations into the virome of humans and other species, only at their very early stages. The development of metagenomics has revolutionised virus discovery, leading to the identification of many new viruses. These studies will generate an enormous amount of data concerning the prevalence of porcine viruses although these will be difficult to interpret, especially with regard to the virus safety of xenotransplantation.
Viral infections are of major concern within the pig production industry, in that they cause not only severe disease but also subclinical infections that can have severe economic consequences. It is becoming evident that a number of factors, including multiple microorganisms, often act synergistically to create a certain clinical picture. This is particularly evident in, for example, complex respiratory and enteric diseases. Known major respiratory viruses include porcine reproductive and respiratory syndrome virus (PRRSV), swine Influenza A virus (swine AIV), and pseudorabies virus (PRV); porcine respiratory coronavirus (PRCV) and porcine circovirus type 2 (PCV2) are also believed to be involved. It is highly plausible that other viruses could be of importance as well, but they have been overlooked. Some reasons for this could be that diagnostic labs are not searching actively for them, because they are not believed to be involved in respiratory diseases, or because they have not even been discovered yet. Additionally, it is known that the combination of viruses and/or bacteria, as well as different management and environmental factors, are of importance. A viral infection that under certain circumstances does not cause any apparent problems for the infected host can, under other circumstances, have severe consequences. Considering this, it is important not only to identify individual viruses in a host but also to investigate the entire viral community.
High-throughput sequencing (HTS) combined with metagenomic approaches has been shown to be a powerful tool for elucidating the aetiology behind diseases, as well as identifying novel viral species from humans, animals, and plants. For pigs, many novel viruses have been identified, including porcine circovirus type 3 (PCV3), porcine bocavirus (PBoV), and atypical porcine pestivirus (APPV). The role that many of these viruses potentially play in disease development is yet to be determined. Apart from detecting previously unrecognised viruses, HTS and metagenomics is a valuable tool for investigating the viral community.
In this study, viral metagenomics was used to characterise the viral community of individual pigs coming from a conventional herd with respiratory lesions at slaughter with the aim of identifying possible agents connected with this disease complex. To understand complex diseases and the role that different viruses may or may not play, we need to identify which viruses are circulating in the porcine population regardless of health status. Thereby, we could possibly identify viruses that make up a basal virome at different ages, in different herds, in connection to different health statuses, etc. This knowledge could be used to understand the effects of different viral-coinfections. In pursuit of this goal, we also investigated the virome of specific pathogen-free (SPF) pigs.
The study did not involve any animal experiment. Tissue samples were collected only from dead animals for laboratory analyses, avoiding unnecessary pain and suffering of the animals. The dog owner gave his written consent for necropsy and sample collection.
Multiple alignments were created using ClustalX (2.0.10). Phylogenetic analyses were carried out with Molecular Evolutionary Genetics Analysis (MEGA), version 5. Picornavirus cleavage sites were predicted by NetPicoRNA 1.0 server (http://www.cbs.dtu.dk/services/NetPicoRNA/) or predicted based on alignments with other picornaviruses. The similarity between different picornaviruses was measured using Simplot version 3.5.1 with the Kimura-2 parameter, a transition/transversion (Ts/Tv) ratio of 3.0 and window and step sizes of 600 and 20 nucleotides, respectively.
For patients with a suspected central nervous system (CNS) infection, rapid and accurate diagnosis is vital to determine treatment and improve prognosis. The differential diagnosis of such patients includes infectious etiologies, of which viruses are the most common, but also non-infectious etiologies, such as auto-immune diseases. Nonetheless, in more than half of cases, the cause remains unknown. Identification of a virus can aid in patient management as it may initiate specific antiviral treatment, or cease or prevent ineffective antiviral, antibiotic, and/or immunosuppressive treatments, which all have potential harmful side effects. For example, when differentiating between an auto-immune and viral origin, immune suppression could lead to deleterious outcomes when caused by an unidentified virus.
During the last two decades, conventional diagnostics for viral CNS infections have shifted from non-specific culturing techniques towards highly-specific viral nucleic acid amplification tests, like quantitative polymerase chain reaction (qPCR), or the detection of host-mediated antibody production to the virus (e.g., ELISA). Although these latter assays have greatly increased diagnostic sensitivity, a limitation is that they only target an individual virus or a subset of related viruses. The number of viruses that have been associated with CNS infections currently comprises more than 100, with several more discovered in the last decade. Consequently, a comprehensive diagnostic panel would include many specific tests. Since this is unachievable for routine diagnostics, only a small selection of viruses commonly associated with CNS infections are included in most diagnostic panels (e.g., herpes simplex virus 1/2, enteroviruses, and parechoviruses). Other pathogens are usually not examined, or are tested for at a later stage of the disease, by which time irreversible pathology could have occurred.
Metagenomics is a recent and promising development in microbiology, which is theoretically able to detect all viruses, including known, unexpected, and novel species. The sensitivity of such assays is generally determined by three factors: (1) The concentration of viruses in a clinical sample, (2) the amount of background (competing) RNA and DNA, and (3) the sequencing depth. Generally, metagenomics assays are poor or unable to detect viruses in a clinical specimen because of the low viral load relative to the high concentration of background RNA and DNA. To overcome this, viral metagenomic assays enrich the viral content of a sample. Virus discovery cDNA-AFLP (amplified fragment length polymorphism) next-generation sequencing (VIDISCA-NGS) is one of the available assays for viral metagenomics. Characteristic for VIDISCA-NGS is the fragmentation of ds(c)DNA, which is done using a frequent-cutting restriction enzyme, and thus different from the random shearing, random PCR amplification, or transposon-based shearing techniques used in most viral metagenomic assays. The method was first described with the discovery of human coronavirus NL63, and since has discovered and detected a wide range of viruses in various sample types. VIDISCA-NGS could be an ideal tool for the broad range detection of viruses in cerebrospinal fluid (CSF).
CSF is a distinct bodily fluid containing a relatively low number of host cells. Even with mild pleiocytosis, as seen during most viral CNS infections, CSF has a far lower cellular content than a similar volume of blood, respiratory, or fecal material. This low amount of background could influence NGS results in two ways: (1) It may decrease the nucleic acid extraction yield if the total nucleic acid content is too low, or (2) it may be beneficial, as proportionally less sequence space is taken by competing background RNA or DNA. Considering the potential benefit viral metagenomics may have for future viral diagnostics in encephalitis, we determined the capability of VIDISCA-NGS to detect viruses in CSF samples from patients with suspected CNS infections.