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Pacific salmon (Oncorhynchus spp.) species have supported coastal ecosystems and Indigenous populations surrounding the North Pacific Ocean for tens of millennia. Today, through their anadromous life history, salmon continue to transport nutrients between aquatic and terrestrial environments (Cederholm et al., 1999), supply the primary food sources for orca whales and sea lions (Wasser et al., 2017; Willson and Halupka, 1995; Chasco et al., 2017; Thomas et al., 2017) and provide economic livelihoods for local communities (Noakes et al., 2002). In the Northeast Pacific, widespread declines of Chinook (O. tshawytscha) and sockeye (O. nerka) salmon have occurred in the last 30 years, leading some populations to the brink of extirpation (Peterman and Dorner, 2012; Heard et al., 2007; Miller et al., 2011; Jeffries et al., 2014), and a cause of great concern to Indigenous groups, commercial and recreational fishers, and the general public. Although the exact number of salmon spawning in rivers is unknown, there are large declines in sockeye salmon over a large geographic area (Peterman and Dorner, 2012). Similarly, Chinook salmon stocks are at only a small percentage of their historic levels, and more than 50 stocks are extinct (Heard et al., 2007).
It is thought that infectious disease may contribute to salmon declines (Miller et al., 2011), but little is known about infectious agents, especially viruses, endemic to Pacific salmon. Infectious disease has been identified as a potential factor in poor early marine survival in migratory salmon; an immune response to viruses has been associated with mortality in wild migratory smolts and adults (Miller et al., 2011; Jeffries et al., 2014), and in unspecified mortalities of salmon in marine net pens in British Columbia (BC) (Miller et al., 2017; Di Cicco et al., 2018). For instance, immune responses to viruses such as Infectious haematopoietic necrosis virus (IHNV) and potentially undiscovered viruses, have been associated with mortality in wild juvenile salmon (Jeffries et al., 2014). This is an important observation as mortality of juvenile salmon can be as high as ~90% transitioning from fresh water to the marine environment (Clark et al., 2016). Together, these suggest that there are undiscovered viruses which may contribute to decreased survival of Pacific salmon but a concerted effort to look for viruses that may contribute to mortality has been absent.
Here, virus-discovery was implemented to screen for viruses associated with mortality. Together, sequencing of dead or moribund aquaculture salmon and live-sampled wild salmon, in-situ hybridization, and epidemiological surveys revealed that previously unknown viruses, some of which are associated with disease, infect wild salmon from different populations.
Figure 1 indicates that two wildlife taxa appear to harbour very few zoonotic pathogens, Manidae (zero) and Hystricidae (one), related to the deficiency of published studies on these taxa, which may lead to an underestimate of their zoonotic infection potential. This lack of data could be attributed to the difficulty of observing these animals in their environment due to their small size and secretive behaviour. Further research is required to determine whether Hystricidae species (Order: Rodentia) harbour more zoonoses, since surveys of other rodents have shown they can host several viruses and bacteria (Easterbrook et al. 2007; Firth et al. 2014).
Globally, one of the most significant threats to wildlife is the overhunting of species for food and commercial gain (Schipper et al. 2008; Maxwell et al. 2016), which is prevalent in the Amazon (Peres 2000), West and Central Africa (Abernethy et al. 2013; Ingram et al. 2015) and Southeast Asia (Bennett et al. 2000; Scheffers et al. 2012; Luskin et al. 2014). The large quantity of wildlife harvested is highlighted in the literature; for example, one study estimated the annual wild meat harvest in the Malaysian state of Sarawak at 23,500 tonnes (Bennett 2002). The increased commercialisation of the wildlife trade facilitates the supply of wild meat to urban consumers (Milner-Gulland and Bennett 2003) and international markets (Chaber et al. 2010). This leads to greater movement of species that increases the likelihood of zoonotic pathogens being translocated, thus presenting health risks to human populations worldwide (Marano et al. 2007). Anthropogenic activities, including the global wildlife trade, have been linked to the rise in emerging infectious diseases (EIDs) (Karesh et al. 2007), and whilst the contribution from the wild meat trade is unknown, its involvement in zoonotic spillovers to humans has been recognised in some countries such as Côte d’Ivoire (Ayouba et al. 2013) and Cameroon (Pernet et al. 2014). “One Health” research (Atlas et al. 2010) synthesises this information and uses collaborative interdisciplinary approaches to improve understanding of zoonotic disease epidemiology in relation to human activities, such as wildlife hunting (Daszak et al. 2007).
People who are involved in wildlife hunting, butchering and consumption risk transmission of infection from their close contact (e.g. transcutaneous, mucosal routes) with live and dead animals or via contaminative routes (e.g. faeces, fomites). Zoonotic infections from hunting are well documented, such as an Ebola disease outbreak related to handling infected chimpanzee, gorilla and duiker carcasses (Leroy et al. 2004) and brucellosis in Australian hunters of wild boar (Eales et al. 2010). Foodborne infections from wild meat consumption have been reported globally, for example, Hepatitis E from raw or undercooked venison in Japan (Matsuda et al. 2003; Tei et al. 2003) and trichinellosis from wild boar meat in France (De Bruyne et al. 2006).
Whilst numerous studies have investigated the zoonotic disease risks from the trade of wild meat in Africa (Wolfe et al. 2005; Kamins et al. 2015), significantly less attention has been focused on Southeast Asia. In this region, many people consume a great variety of wildlife due to their cultural practices and beliefs. The demand for species valued as a delicacy, such as Sumatran serow meat in Malaysia (Shepherd and Krishnasamy 2014), or used for traditional medicine, including Asiatic softshell turtles in soup (Sharma 1999), has led to greater commercialisation of the trade within Southeast Asia (Scheffers et al. 2012; Shepherd and Krishnasamy 2014), which increases risks for human health. Since the wildlife trade distribution networks enable the regional movement of animals, this facilitates cross-species transmission of pathogens due to the mixing of numerous species from different sources in combination with the close proximity between wildlife and humans (Karesh et al. 2005). The importance of understanding how these networks influence zoonotic infection between species was illustrated by the spread of severe acute respiratory syndrome (SARS)-associated coronavirus from bats to civets to humans (Li et al. 2005c).
This aim of this review is to fill the gap in knowledge about Southeast Asia by evaluating published research to determine the potential zoonotic infection risks to humans from hunting, butchering and consumption of wildlife, using the wild meat trade in Malaysia as a case study.
Fish were screened against a viral disease detection biomarker panel (VDD) that elucidates a conserved transcriptional pattern indicative of an immune response to active RNA viral infection (Miller et al., 2017). For instance, in a previous study, we showed that 31% of moribund Atlantic salmon were in a viral disease state, and half of these were not known to be positive for any known RNA viruses (Di Cicco et al., 2018). Individuals that were strongly VDD-positive, but negative for any known salmon viruses (e.g. Piscine orthoreovirus, Erythrocytic necrosis virus, Infectious pancreatic necrosis virus, Infectious hematopoietic necrosis virus, Infectious salmon anaemia virus and Pacific salmon paramyxovirus) were subject to metatranscriptomic sequencing. The sequencing revealed viral transcripts belonging to members of the Arenaviridae, Nidovirales and Reoviridae, three evolutionarily divergent groups of RNA viruses (Figure 1) that can be highly pathogenic (Yun and Walker, 2012; Liang et al., 2014; Weiss and Leibowitz, 2011).
One of the challenges of viral discovery in fish is that the proportion of viral transcripts in vertebrate metatranscriptomic libraries is small compared to the number of transcripts from the host and other contaminating sequences (Geoghegan et al., 2018; Zhang et al., 2019). However, we were able to achieve near-coding complete genomes for the three new viruses (Figure 1—figure supplement 1A and B). The genomic organisation of the newly discovered viruses was consistent with related viruses in fish. For instance, SPAV has three genomic segments, as shown for other arenaviruses in fish (Shi et al., 2018). High-throughput RT-PCR screening of >6000 wild juvenile Chinook and sockeye salmon showed dissimilar geographical distributions of infected fish, reflecting differences in epidemiological patterns of transmission and infection dynamics for each of the viruses (Figure 2).
The distribution and abundance of the different viruses varied markedly. Arenaviruses were relatively common (Figure 2—figure supplement 1) and geographically widespread in migratory juvenile Chinook and sockeye salmon in the marine environment (Figure 2, Figure 2—figure supplement 2). Whereas, the nidovirus was spatially localised and predominantly observed at high prevalence over multiple years in Chinook salmon leaving freshwater hatcheries (Figure 2). Finally, the reovirus was detected only in farmed Chinook salmon (Figure 2 and Figure 2—figure supplement 1).
With the exception of their relatively recent discovery in snakes (Stenglein et al., 2012) and frogfish (Shi et al., 2018), arenaviruses were thought to solely infect mammals. The arenaviruses reported here share less than 15% amino-acid sequence similarity (in the RdRp) to those from mammals and snakes, and define a new monophyletic evolutionary group, the pescarenaviruses (Figure 1A). The absence of clear sequence homology in the glycoprotein, the difference in genome segmentation (Shi et al., 2018), as well as phylogenetic analysis of the replicase demonstrate that pescarenaviruses share a common but ancient ancestor with arenaviruses infecting snakes and mammals. We recommend these fish-infecting arenaviruses are assigned to the new genus Pescarenavirus, with those infecting Chinook and sockeye salmon being assigned to the species Salmon pescarenavirus (SPAV), strains 1 and 2, respectively.
Farmed Chinook salmon positive for SPAV-1 displayed pathology and symptoms consistent with disease including inflammation of the spleen and liver, as well as tubule necrosis and hyperplasia in the kidney. Clinically, salmon presented with yellow fluid on the pyloric caeca and swim bladder, pale gills with haemorrhaging on the surface, and anaemia. Wild Chinook and sockeye that tested positive for arenavirus infection, but which were clinically healthy when sampled, showed few histological lesions. In-situ hybridization revealed that arenaviruses were concentrated mainly in macrophage-like cells, melanomacrophages, red-blood cells (RBCs) and endotheliocytes (Figure 3). These findings are consistent with localisation of arenaviruses in mammals and snakes, although in contrast to snakes and fish, mammalian red blood cells are not nucleated so the similarity likely only extends to nucleated cells. SPAV-1 and −2 shared similar cell tropism within Chinook and sockeye salmon, respectively (Figure 3—figure supplement 1). In one out of the eight Chinook samples examined, moderate chronic-active hepatitis was reported, and staining for SPAV-1 was identified in the area affected by inflammation (Figure 3C and D), while in the other samples SPAV-1 was confined to reticuloendothelial cells in the liver tissue or in the sinusoids. More lesions were observed in dead farmed Chinook, where disease progression is more advanced. Our observations indicate that arenaviruses are replicating in red-blood cells, and occur in the macrophages and leukocytes that consume the infected cells. Moreover, the observed pathological changes in arenavirus-infected fish, including anaemia, and lesions in the gills, kidney and liver would be expected for viruses that infect red-blood cells. These results are the first empirical evidence for arenavirus infection in fish, and suggest that SPAV, like many other arenaviruses, has the potential to be a causative agent of disease.
Sequencing of cultured Chinook salmon also revealed a previously undescribed nidovirus and reovirus. Phylogenetic analysis of the reovirus, named Chinook aquareovirus (CAV), predicts that it is part of the genus, Aquareovirus (Figure 1B). Rather than being most closely related to known reoviruses of salmon (Winton et al., 1981), CAV groups with a growing number of aquareoviruses, some of which are known to cause haemorrhagic disease and have led to serious losses to aquaculture in China (Nibert and Duncan, 2013; Wang et al., 2012). The observed clinical signs (anemia, dark spleen, and blood-filled kidneys) in dead farmed Chinook salmon with high loads of CAV are consistent with a haemorrhagic manifestation.
The novel nidovirus, named Pacific salmon nidovirus (PsNV), is most closely related to the recently described Microhyla alphaletovirus 1, which forms a sister group to the coronaviruses (Bukhari et al., 2018). This sequence, alongside PsNV are basal to all other Nidovirus families, and their long branch length suggests they each belong to a different genus (Figure 1C). While not all coronaviruses cause serious disease, many do, such as SARS and MERS, which cause severe respiratory infections (de Wit et al., 2016).
Both SPAV-1 and SPAV-2 were relatively widespread along the coast of southwestern British Columbia, in ocean caught Chinook and sockeye salmon. Currently, it is unclear what is driving differences in SPAV-1 and SPAV-2 prevalence among regions, but the virus appears to be transmitted to juvenile salmon throughout southern BC soon after they enter the ocean, a period known to be critical to their survival (Beamish et al., 2012a). SPAV-1 was also relatively common in farmed Chinook populations. The distribution of SPAV-1 in wild Chinook populations was more localised to the west coast of Vancouver Island than SPAV-2, which was most prevalent on the east coast of Vancouver Island, near the Discovery Islands and the Johnstone Strait, and was rarely detected in sockeye salmon in northern BC and Alaska (Figure 2—figure supplement 2).
On the east coast of Vancouver Island, the Johnstone Strait and Discovery Islands have been identified as a potential choke point for the growth and survival of juvenile salmonids (Healy et al., 2017). The availability of prey to juvenile sockeye in the northern Johnstone Strait is extremely low, resulting in food limitation and increased competition for prey (Beamish et al., 2012a; McKinnell et al., 2014; Godwin et al., 2015; Godwin et al., 2018). These regions of high SPAV-2 infection could represent a stressful part of juvenile sockeye outmigration, possibly resulting in higher susceptibility to infection. Moreover, SPAV-2 was detected at high loads in fish sampled from regions where finfish aquaculture facilities are abundant and accordingly, sea lice infestation is high (Price et al., 2011]. It remains an open question whether an alternative host could play a role in virus transmission between fish, and/or result in an increased susceptibility to infection (Valdes-Donoso et al., 2013).
The distribution of CAV was markedly different from SPAV. CAV was not detected in any juvenile wild or hatchery Chinook salmon, despite being detected in farmed fish on both the west and east coasts of Vancouver Island. Over 20% of moribund Chinook aquaculture fish tested positive for CAV, with most detections occurring in fish at least 1.5 years after ocean entry, well past the time when migratory salmon were sampled. Hence, infection by CAV may take a considerable time to develop, or be an infection that is only acquired by older fish. CAV was also detected in a small number of farmed Atlantic salmon (seven positive detections of 2816 fish tested). The monophyletic grouping of CAV with other disease causing aquareoviruses and the consistency with haemorrhagic disease suggest that the virus is important to monitor in cultured fish, and potentially wild adults returning after several years at sea.
PsNV distribution was strongly associated with a handful of salmon-enhancement hatcheries but was also detected in 18% of aquaculture Chinook and 3% of wild Chinook (Figure 2—figure supplement 1). In hatchery fish, infection by PsNV was primarily localised to gill tissue (Figure 4A), reminiscent of the respiratory disease caused by the related mammalian coronaviruses such as MERS and SARS (Figure 1C). PsNV is of particular concern as it proliferates while fish are undergoing smoltification, a process during which the gill tissue undergoes cellular reconfiguration to prepare for saltwater. Notably, branchial proliferation of no known cause was noted in some farmed salmon infected with PsNV. In one of the hatcheries, where pre- and post-release sampling took place, the virus increased in prevalence during smolt development in fresh water, was detected shortly post-release, and was barely detected in the month following ocean entry (Figure 4B). This suggests that infected fish either cleared the infection, or did not survive after entry into the marine environment. The second interpretation is consistent with the lower rates of ocean survival in fish produced from hatcheries versus wild salmon (Beamish et al., 2012b).
Viral disease is a potential threat to wild fish stocks; yet little is known about viruses circulating in wild, farmed, or hatchery salmon. Here, through metatranscriptomic surveys, we reveal several previously unknown viruses that were discovered in dead and dying aquaculture fish, and show them to also occur in wild and hatchery-reared fish. Depending on the viral and host species, the viruses range from being localised to widespread, from infecting <1% to >20% of fish, and being from within the limits of detection to very high loads. Our results are consistent with some of these viruses being causative agents of disease, making it critical to understand their possible roles in salmon mortality and the decline of wild salmon populations, and their potential interactions with net-pen fish farming and hatchery rearing. Viral discovery in moribund individuals followed by extensive surveillance and histopathological localisation are powerful tools towards the ultimate goals of identifying causative agents of disease and understanding the impact of infectious agents in wild populations. These insights are crucial as juvenile salmon that are in less than optimal health are expected to have lower rates of survival in the wild. Continued surveillance and knowledge of endemic and emerging virus infections in these iconic salmon species is beneficial for their conservation.
Olive flounder is one of main marine species having high economic value in the countries of East Asia. Because of industrial importance by increasing of demand, it is serious subject to understand the pathogenic infection and production performance of olive flounder. Mass mortality of fishes is the most severe problem, accompanying vast deficit in aquaculture farm. Efforts to prevent regrettable death have especially been conducted in immunology1–3. However, so far, it is hard to figure out cause of death exactly in short time because of variables of expansive marine ecosystem. Mass mortality of olive flounder was normally caused by diseases via various sources of infection such as virus, bacteria, or parasite from external environment.
Viruses depend on host cell ribosomes to produce their proteins, and sometime use host cell DNA and RNA polymerases for replication and transcription, respectively. Many viruses encode proteins that modify the host transcription or translation apparatus to favor the synthesis of viral proteins over those of the host cell. Among viruses, viral hemorrhagic septicemia virus (VHSV) is affiliated to Novirhabdovirus genus, which is a member of the Rhabdoviridae family4. The six gene were contained in the VHSV genome of about 11 K bases and each of them coded nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), nonstructural viral protein (NV), and RNA polymerase (L) in the following order 3’-N-P-M-G-NV-L-5’4. Infection of VHSV results in contagious viral hemorrhagic septicemia (VHS) in diverse fish species regardless of their inhabitation; seawater or freshwater5. In East Asia, a lot of infection cases into olive flounder have been reported steadily, since VHSV was detected in middle of 1990s6–9.
A variety of scuticociliates have been reported as cause of scuticociliatosis in marine species including turbot, guppy, and southern bluefin tuna10–12. In olive flounder, disease has been reported to be causing from various scuticociliates; Uronema marinum, Pseudocohnilembus persalinus, Philasterides dicentrarchi, Miamiensis avidus13–16. Interestingly, judging from infection experiments using various scuticociliates plus identification outcome of 8 isolates acquired from olive flounders with symptom of ulcers and haemorrhages, Miamiensis avidus was suggested as the major aetiologic agent of scuticociliatosis because of high pathogenicity and mortality rate compared with other scuticociliates14,17.
Infection of bacteria could sustain serious damage to fish. Streptococcosis is known to be caused by a variety of streptococcic species; Streptococcus parauberis, Streptococcus iniae, Streptococcus difficilis, Lactococcus garvieae, Lactococcus piscium, Vagococcus salmoninarum, and Carnobacterium piscicola, and has become major nuisance in olive flounder farms18–21. In particular, Streptococcus iniae, Lactococcus garvieae, and Streptococcus parauberis have been introduced to be related with Streptococcosis in olive flounder19–23.
The main issue of aquaculture industry is to reduce economic loss by preventing mortality of fish from various pathogens. A large number of immunologic studies have been proceeded about various immune-related gens against pathogen infection3,24–27. A huge quantity of genomic information from next generation sequencing (NGS) technique has been gradually increasing for the last few years, indicating that researchers could approach more comprehensive understanding view about genome of organisms than when they research a single gene level. With development of wide-sized analysis methods, it is not difficult to figure out change of gene expression level after any chemical treatment or environmental change. Recently, studies to identify large-scale genes were conducted in the olive flounder genome for researches about vaccine, gonadal development, and sex determination28–30. In particular, characterizing of immune-related genes was reported in olive flounder spleen tissue31. A lot of studies reported earlier were focused on gene expression analysis of single pathogen and specifically defined the expression pattern of limited genes32–36. Further, infection by two or more pathogens were reported in the olive flounder genome37,38. In order to solve these problem, we need plentiful genomic information to respond rapidly to multiple infection of pathogens. However, researches, which were comprehensively analysed about change of gene expression pattern by different type of pathogens, have not been reported in the olive flounder genome, so far.
In this research, we identified differentially expressed genes (DEGs) by transcriptome analysis and conducted gene ontology (GO) analysis with genes identified. Then, we tried to find important genes which showed consistently meaningful expression change in the results of three infection experiments. As a result, we determined 10 up-regulated genes and 57 down- regulated genes in common after infection of three pathogens. We aimed to provide essential genome information which is related with pathogen infection and explore the various consequences related to differential infections and find out the common strategies against specific candidates involved in disease progression in natural habitat of aquaculture.
Peste des petits ruminants (PPR), also known as ovine rinderpest or goat plague, is an acute, highly contagious viral disease of goats and sheep, caused by the Peste des petits ruminants virus (PPRV), a morbillivirus in the family Paramyxoviridae. The disease is characterized by high fever, nasal and ocular discharges, pneumonia, necrotic and ulcerative lesions of the mucus membranes and inflammation of the gastro-intestinal tract. PPRV infection results in great economic losses and affects productivity of sheep and goats subsequent to the global eradication of Rinderpest. For example, in 2004, the economic cost of PPRV in India was estimated to be 1800 million Indian rupees (US$ 39 million) per year,. PPRV replication and seroconversion has been demonstrated in large ruminants. There is a solitary report on clinical PPRV occurring in water buffalo, although it has not been confirmed in later studies. In September 2004, outbreaks of PPR in Sudan affected both sheep and camels.
PPR is generally considered a more serious disease in goats than sheep, however, increased susceptibility of sheep, goat and outbreaks involving both sheep and goats have been equally reported,,,,. Goats appear not to be affected in some outbreaks, while sheep suffer with high rates of mortality and morbidity. Strain specific virulence of PPRV has been reported when the same breed of goats were experimentally infected, and different breeds of goat have been shown to respond differently to infection with the same virus. Species-specific disease occurrence has been observed with foot and mouth disease, where cattle were highly affected while sheep had less severe infection with the virus. Epizootic haemorrhagic disease virus affects cattle but sheep do not suffer from this disease. It is well recognised that ducks were generally resistant to avian influenza virus (AIV) whereas chickens suffer from severe disease with rapid death following infection with highly pathogenic AIV. The reason for this species specificity is unclear at present.
The natural susceptibility to PPRV in goats could be attributed to several host-derived or virus-derived factors. One such host-derived factor could be the differential presence or distribution of specific viral receptors in these species, such as the signalling lymphocyte activation molecule (SLAM) that has previously been observed to be associated with PPRV and other morbilli viruses such as measles virus and canine distemper virus,,. Host immune mechanisms could also account for this differential susceptibility, although this has not been explored in detail in ruminant species or breeds.
Toll like receptors (TLR) are type 1 transmembrane proteins expressed in almost all cell types and activate the innate immune system upon sensing pathogen associated molecular patterns (PAMPs). Intracellular TLR that sense viral nucleic acids include TLR3 (double stranded RNA), TLR7 and TLR8 (single stranded RNA) and TLR9 (CpG motifs in DNA). Imiquimod and poly I:C are standard agonists used to induce TLR7 and TLR3 respectively leading to the production of inflammatory cytokines including type I interferons (IFN) and immune cell maturation,. TLR are differentially expressed in various tissues and immune cells of water buffalo and goats, and have been shown to induce differential immune responses,. The cell specific location and basal expression levels of TLR mRNA could indicate the natural PAMP load of that tissue as well the innate host resistance to pathogens.
In addition to the differential expression profiles of TLR, ligand induced downstream cytokine profiles and/or levels could also play a role in the innate disease resistance of a species or breed. For example, mastitis is an economically important inflammatory disease of the udder that has been shown to be more prevalent in the Holstein breed of cows than in Jersey cows. This has been linked to temporal differences in the onset and duration of immune responses, including cytokines such as tumor necrosis factor alpha (TNFα) and IFNγ, following intramammary inoculation of pathogenic E. coli.
India has 23 genetically well characterized indigenous breeds of goats (http://www.icar.org.in/en/node/4688). Barbari is a common breed of goat reared for meat and milk production in northern India. Tellicherry, Kanni and Salem black are indigenous breeds of goats prevalent in southern India. Outbreaks of PPR have been reported in newly introduced Barbari goats to southern India with mortality rates of 16.67% to 65.0%. Recently, a severe outbreak of PPR was reported in Tellicherry breed of goats with 100% mortality in kids and 87.5% mortality in adults. PPRV infection appears to be subclinical in Kanni and Salem black breeds of goats. Similarly, native water buffalo in India are resistant to PPRV. Although such anecdotal evidence and field observations on differential resistance within goat breeds and water buffalo are available, experimental evidence is lacking for the observed differences in susceptibility.
We hypothesized that the differential susceptibility of various Indian goat breeds and native water buffalo to PPRV could be related to innate immune resistance mechanisms. Infection of peripheral blood mononuclear cells (PBMC) from four breeds of goats and water buffalo resulted in differential viral replication kinetics and inflammatory cytokine profile including IFNα, IFNγ and TNFα with differential activation of TLR3 and TLR7. Analysis of single nucleotide polymorphisms (SNPs) in the complete gene sequences of TLR7 between goat breeds did not show any differences that could account for this.
Coronaviruses (CoV) are the causative agents of significant diseases resulting in substantial impact on human and animal health. Both severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) have caused a significant burden on human health, including a number of deaths, and have had significant socioeconomic impacts on the countries in which people were infected1–3. Coronavirus infections of livestock, such as porcine epidemic diarrhoea virus (PEDV) and more recently porcine deltacoronavirus (PDCoV) in pigs and infectious bronchitis virus (IBV) and turkey coronavirus in poultry, have significant impacts on animal health and cause considerable economic costs to producers4–7.
Interspecies spill-over of coronaviruses into new hosts occurs frequently, with SARS-CoV and MERS-CoV being the most notable examples of spill-over into humans8–10. Bovine coronavirus, canine respiratory coronavirus, dromedary camel coronavirus and even human coronavirus OC43 all potentially come from the same common ancestor, illustrating substantive host flexibility11–13. SARS-CoV likely originated in bats while PDCoV interestingly is likely to have originated in birds6,8,9. Consequently, there is significant interest in assessing wild animals for CoV’s.
Wild birds are ubiquitous and highly mobile potential hosts capable of moving viruses over large distances and across geographical and political borders. Wild birds have been implicated in the spread of highly pathogenic H5Nx avian influenza viruses14 and bird migration patterns describe phylogenetic patterns in the matrix gene of low pathogenic influenza virus15. Coronaviruses have been detected in a range of species of wild birds on all continents except for Australia and Antarctica16–21. It is likely that CoV’s are present in wild birds on all continents. However, a recent survey in Australia of 409 birds failed to detect coronaviruses22.
Studies in which wild bird coronaviruses have been successfully detected, have commonly sampled aquatic bird species such as Anseriformes (ducks, geese and swans) and Charadriiformes (gulls, migratory shorebirds and waders)16,18,20,23,24. This prompted our study to look for coronaviruses in Australian birds that notably belong to these two orders.
Given that the genetic diversity of CoV’s in Australian wild birds was unknown it was decided to use a combination of PCR assays to maximise the probability of detecting positive individuals. Coronavirus positive samples were genetically sequenced and phylogenetic analysis performed to compare these sequences to coronavirus sequences from previous surveys of wild and domestic birds across the globe.
Small volant and nonvolant mammals are important components of ecological communities and play vital roles in ecological systems. They are among the most common agents for infections and, thus, have strongly affected human history. For example, black rats (Rattus rattus) are considered likely agents for the spread of Oriental rat fleas, which drove the Black Death plague throughout Europe and the Mediterranean during the 14th century and killed 30%–60% of the European population (Barnett, 2001; Duplantier et al., 2003). More recent examples of small mammal zoonoses include severe acute respiratory syndrome (SARS) caused by a coronavirus and Ebola hemorrhagic fever caused by Ebolavirus, with hosts including, but not limited to, bats and civets (Klein & Calisher, 2007; Menachery et al., 2015). Rodent-borne diseases such as plague and hantavirus have made considerable contributions to human illnesses and are responsible for more deaths than all wars combined (Klein & Calisher, 2007). New pathogens, especially hantaviruses, have been isolated from rodents in China and adjacent countries annually (Huang et al., 2017). Because different species have specific immune systems and different levels of tolerance to zoonotic infections, identification of rodent reservoirs of zoonotic pathogens is a high priority (Meerburg et al., 2009).
Rats and mice often top the zoonoses reservoir list of the Chinese Center for Disease Control and Prevention (China CDC) because of the large number of species, substantial population sizes, and high potential for carrying zoonotic pathogens (Wu et al., 2017). Unfortunately, we still do not know how many species of rats and mice occur in China, or which species carry what pathogens, even for the most common genera such as Apodemus and Rattus. The reasons for this are complicated. Both Apodemus and Rattus have complex evolutionary and taxonomic histories, with classifications continuously being updated. Switching between valid species and synonyms causes considerable confusion, especially for non-specialist researchers. Furthermore, many species occur only in remote mountains or near national borders with high species diversity, such as Yunnan, Xizang (Tibet), and Xinjiang. Indeed, the rats and mice of southern Xizang and western Xinjiang remain to be studied carefully. Finally, many rodents are difficult to identify to species level due to the number of morphologically similar species (Galan et al., 2012).
The latest version of Mammal Species of the World (Musser & Carleton, 2005) recognized 20 species of Apodemus and 162 synonyms. Several scenarios for the classification of Apodemus have been proposed (Filippucci, 1992; Martin et al., 2000; Musser et al., 1995; Serizawa et al., 2000; Zimmermann, 1962), but none are strongly supported, and phylogenies remain poorly resolved despite molecular efforts (Liu et al., 2004; Serizawa et al., 2000; Suzuki et al., 2003). Furthermore, the number of species in China remains unknown, with previous estimations varying from six (Corbet, 1978; Xia, 1984), seven (Liu et al., 2002; Liu et al., 2004), eight (Smith et al., 2008), and nine species (Nowak, 1999; Wang, 2003). Many authors have suggested that A. sylvaticus occurs in Xinjiang, China (Corbet, 1978; Xia, 1984; Wang, 2003), whereas others have argued that the species is A. uralensis (Smith et al., 2008). The former species occurs in Western Europe (Bousbouras, 1999; Macholán et al., 2001; Mezhzherin & Zykov, 1991; Michaux et al., 1996), and its incorrect identification in China likely relates to outdated taxonomy.
Rattus, another problematic genus, has had 25 subgenera and more than 550 species and subspecies named (Simpson, 1945). Currently, 66 species are recognized but uncertainty persists. Previous supermatrix analysis did not obtain a monophyletic Rattus, indicating that systematics is far from resolved (Steppan & Schenk, 2017). Arguments also persist for the most common species, including black rats whose species boundary remains unfixed (Aplin et al., 2011). The number of species of Rattus in China is also uncertain and varies from four (Corbet, 1978), seven (Smith et al., 2008), and nine (Wang, 2003).
Similar to other rodents, species in these two genera are difficult to identify or distinguish morphologically due to their similar appearance, overlapping measurements, and key factors involving the single cusp on their teeth. Diagnosis often requires clean skulls, which are not always available or correctly prepared. DNA barcoding is a promising approach but requires a solid reference database (Moritz & Cicero, 2004). Unfortunately, GenBank data are problematic because many rodent sequences are uploaded by non-specialists such as epidemiological researchers. This reduces the reliability of environmental assessment reports and hampers our understanding of host and disease associations.
Herein, we revisited the alpha diversity of Apodemus and Rattus in China based on a collection of more than 400 specimens and the integration of cyt b sequences. We evaluated the species of both genera in China and assessed if they could be identified easily using traditional morphometric approaches.
Middle East respiratory syndrome coronavirus (MERS-CoV), endemic in camels in the Arabian Peninsula, is the causative agent of zoonotic infections and limited outbreaks in humans. The virus, first discovered in 2012 (Zaki et al., 2012; van Boheemen et al., 2012), has caused more than 2000 infections and over 700 deaths, according to the World Health Organization (WHO) (World Health Organization, 2017). Its epidemiology remains obscure, largely because infections are observed among the most severely affected individuals, such as older males with comorbidities (Assiri et al., 2013a; WHO MERS-Cov Research Group, 2013). While contact with camels is often reported, other patients do not recall contact with any livestock, suggesting an unobserved community contribution to the outbreak (WHO MERS-Cov Research Group, 2013). Previous studies on MERS-CoV epidemiology have used serology to identify factors associated with MERS-CoV exposure in potential risk groups (Reusken et al., 2015; Reusken et al., 2013). Such data have shown high seroprevalence in camels (Müller et al., 2014; Corman et al., 2014; Chu et al., 2014; Reusken et al., 2013; Reusken et al., 2014) and evidence of contact with MERS-CoV in workers with occupational exposure to camels (Reusken et al., 2015; Müller et al., 2015). Separately, epidemiological modelling approaches have been used to look at incidence reports through time, space and across hosts (Cauchemez et al., 2016).
Although such epidemiological approaches yield important clues about exposure patterns and potential for larger outbreaks, much inevitably remains opaque to such approaches due to difficulties in linking cases into transmission clusters in the absence of detailed information. Where sequence data are relatively cheap to produce, genomic epidemiological approaches can fill this critical gap in outbreak scenarios (Liu et al., 2013; Gire et al., 2014; Grubaugh et al., 2017). These data often yield a highly detailed picture of an epidemic when complete genome sequencing is performed consistently and appropriate metadata collected (Dudas et al., 2017). Sequence data can help discriminate between multiple and single source scenarios (Gire et al., 2014; Quick et al., 2015), which are fundamental to quantifying risk (Grubaugh et al., 2017). Sequencing MERS-CoV has been performed as part of initial attempts to link human infections with the camel reservoir (Memish et al., 2014), nosocomial outbreak investigations (Assiri et al., 2013b) and routine surveillance (Wernery et al., 2015). A large portion of MERS-CoV sequences come from outbreaks within hospitals, where sequence data have been used to determine whether infections were isolated introductions or were part of a larger hospital-associated outbreak (Fagbo et al., 2015). Similar studies on MERS-CoV have taken place at broader geographic scales, such as cities (Cotten et al., 2013).
It is widely accepted that recorded human MERS-CoV infections are a result of at least several introductions of the virus into humans (Cotten et al., 2013) and that contact with camels is a major risk factor for developing MERS, per WHO guidelines (World Health Organization, 2016). Previous studies attempting to quantify the actual number of spillover infections have either relied on case-based epidemiological approaches (Cauchemez et al., 2016) or employed methods agnostic to signals of population structure within sequence data (Zhang et al., 2016). Here, we use a dataset of 274 MERS-CoV genomes to investigate transmission patterns of the virus between humans and camels.
Here, we use an explicit model of metapopulation structure and migration between discrete subpopulations, referred to here as demes (Vaughan et al., 2014), derived from the structured coalescent (Notohara, 1990). Unlike approaches that model host species as a discrete phylogenetic trait of the virus using continuous-time Markov processes (or simpler, parsimony based, approaches) (Faria et al., 2013; Lycett et al., 2016), population structure models explicitly incorporate contrasts in deme effective population sizes and migration between demes. By estimating independent coalescence rates for MERS-CoV in humans and camels, as well as migration patterns between the two demes, we show that long-term viral evolution of MERS-CoV occurs exclusively in camels. Our results suggest that spillover events into humans are seasonal and might be associated with the calving season in camels. However, we find that MERS-CoV, once introduced into humans, follows transient transmission chains that soon abate. Using Monte Carlo simulations we show that R0 for MERS-CoV circulating in humans is much lower than the epidemic threshold of 1.0 and that correspondingly the virus has been introduced into humans hundreds of times.
The grass carp (Ctenopharyngodon idellus) is one of the most famous aquaculture species worldwide, accounting for 13% of global freshwater aquaculture production in 2014 [1, 2]. However, frequent diseases always result in huge economic loss to the grass carp cultivation industry. Of these diseases, grass carp hemorrhage disease caused by grass carp reovirus (GCRV) has been being noticed with special concern by fish breeding scientists with the hope of disease-resistant breeding. GCRV, belonging to the family Reoviridae, genus Aquareovirus, is a double-stranded RNA (dsRNA) virus reported mainly in China and could trigger apoptosis in C. idellus kidney (CIK) cells. However, the mechanism of apoptosis induced by GCRV, which is critical for the defense of GCRV and virus-resistant breeding, remains unknown. The rare minnow (Gobiocypris rarus), a Chinese native species belonging to the family Cyprinidae, could be infected by GCRV, resulting in an up to 100% mortality rate. Moreover, due to its propagational biological characteristics, rare minnow has the potential to be used as a model fish in aquatic toxicity testing, chemical safety assessment, and virus-resistant breeding.
In CIK cells, both death receptor pathway and mitochondrial pathway were involved in GCRV-induced apoptosis. Bid, BH3-interacting domain death agonist, is a pro-apoptotic BH3-only member of Bcl-2 family, serving as a key link in the amplification of various apoptosis signals other than the death receptor pathway [7, 8]. In the mitochondrial pathway, caspase-8 activated by death receptors pathway cleaves Bid and the truncated Bid (tBid) then translocates to the mitochondria. The BH3 domain of tBid can interact with pro-apoptotic Bcl-2 proteins, including Bax and Bak, to mediate the mitochondrial outer membrane permeabilization (MOMP), leading the release of cytochrome c (cyt c) into cytosol with subsequent activation of apoptosis [7–13].
Interestingly, numerous studies in animal models demonstrated that Bid could be viewed as potential therapeutic target for some certain diseases. Suppression of hepatocyte Bid using an antisense approach could not only effectively attenuate the hepatocytes apoptosis induced by Glychochenodeoxycholate (GCDC), but also ameliorate liver injury in a mice model of extrahepatic cholestasis. In hepatocellular-specific Bid deficient mice, their liver showed strong resistance to hepatocellular apoptosis and hepatotoxicity in comparison with controls, suggesting inhibition of Bid was critical for the resistance to the lethal effects of Fas activation in vivo. In contrast to controls, an upregulation of Bid could be detected in septic shock patients, indicating pro-apoptotic gene Bid has great potential as a biomarker to monitor sepsis. Bid-/- mice showed no lung injury after Lipopolysaccharides (LPS) stimulation. Survival of Bid-deficient mice was significantly increased when compared with wild type mice after reovirus infection [7, 18]. However, there is limited knowledge of Bid in teleost fish, and the specific role of Bid during the virus-induced apoptosis in teleost fish is still unclear.
This study examined the mechanisms of GCRV-induced apoptosis and investigated the possibility that knocking out of Bid may provide an innovative strategy for enhancing survival of rare minnow following virus infection. In the present study, Bid genes from grass carp (CiBid) and rare minnow (GrBid) were cloned and analyzed. The expression pattern of CiBid among different tissues and the response to GCRV were studied in vivo. Furthermore, we examined the function of Bid using genetically deficient (Bid-/-) rare minnow. Our results suggested that the replication of GCRV and virus-triggered apoptosis were both suppressed in Bid-/- rare minnow in comparison with wild-type rare minnow. Our study provides new insight into understanding the GCRV induced apoptosis and suggests that Bid may be a target gene for virus-resistant breeding in grass carp.
To determine if treatment with BTE interfered with viral adsorption in A549 and Vero cells, either in part or in whole, four assays were performed and compared to an untreated sample infected by HSV-1. In the plaque reduction assay a slight reduction in plaques was observed in the BTE treated group (Figure 6A), possibly due to the inhibitory effects of BTE previously mentioned. The virus adsorption assay (Figure 6B) showed a reduced number of plaques in the BTE treated sample, indicating that some part of adsorption was affected. Two additional assays were performed to further assess which aspect of viral adsorption was affected. The virus attachment assay displayed a significant reduction in the plaques formed in the BTE treated group (Figure 6C), while the penetration assay showed a similar reduction in plaque formation in the BTE treated group (Figure 6D).
Black grain eumycetoma represents the most common fungal mycetoma worldwide. This chronic, erosive infection of subcutaneous tissues particularly affects the lower extremities and leads to severe disability. The disease is considered a major health problem in tropical areas and is prevalent among people of low socio-economic status.
Mycetoma presents as a subcutaneous mass with multiple sinuses that discharge pus, serous fluid and grains, i.e. the characteristic compact grains of the causative agent formed inside the lesion.
A wide range of microorganisms has been reported to cause mycetoma. For treatment, not only differentiation between (fungal) eumycetoma and (bacterial) actinomycetoma is important, but also the identity of the causative agent, since species differ in their response to antimicrobial drugs. In endemic countries, clinical diagnosis may be the only diagnostic method. A fully developed mycetoma lesion is easily identified clinically, whereas in early stages with the absence of grains, the infection may be confused with phaeomycosis or soft tissue tumors. In such cases fine needle aspiration cytology or deep surgical biopsy for histological examination are useful,. Some fungal and bacterial grains have a characteristic histological appearance which helps in provisional identification, but recognition of the causative species remains impossible. Isolation of the pathogen from discharged grains or from biopsies allows identification of agents that sporulate, but most of the species lack phenotypic characteristics. Molecular techniques have been introduced to facilitate the identification of nondescript organisms,,, but are of high cost and time-consuming. Thus, there is a need for a fast, simple and reliable method for identification.
Rolling circle amplification (RCA) is a powerful diagnostic method based on detection of specific nucleic-acid sequences and enzymatic amplification of circularized oligonucleotide probes under isothermal conditions. The probes are linear oligonucleotides that contain two target-complementary sequences at their ends joined by linkers. The ends of the probe hybridize to the complementary target in juxtaposition and then ligate which allows the circularization of the probe. The circular structured molecule then amplifies with DNA polymerase that has strand displacement and progressive DNA synthesis activity resulting in series of repeats of the original circular template,. The technique has been proven to be rapid, specific and low-cost for molecular identification of viruses, bacteria, and fungi,,,. It has been applied for identification of a rare black grain mycetoma species Exophiala jeanselmei
. In addition, RCA has been used successfully for identification of white grain mycetoma species Scedosporium boydii
. The aim of the present study is to develop RCA-based diagnostics for the most common agents of black-grain eumycetoma.
To confirm the findings of phase contrast microcopy and the plaque assay, fluorescent microscopy (400×) was employed to visually examine progeny virions in cells that were exposed to HSV-1 treated with 1.4 mM of BTE. For A549 samples, at 12 hours post-infection, there was a pronounced fluorescence from cells infected with untreated HSV-1 (Figure 3A), yet no viral fluorescence was detected from either the control (cells treated with 10% FBS-media) (data not shown) or cells inoculated with HSV-1 treated with BTE (Figure 3B). At 24 hours post-infection, there was still a significant amount of fluorescence from cells infected with untreated HSV-1 (Figure 3C), but only a small amount of fluorescence from cells inoculated with HSV-1 treated with BTE (Figure 3D). For Vero cells infected with untreated HSV-1, there was a significant amount of fluorescence 36 hours post-infection; Vero cells infected with increasingly higher concentrations of BTE showed decreasing levels of fluorescence (Figure 4).
PCR amplification of BTE-treated HSV-1 infected A549 and Vero cells indicates that the replication of viral genes for glycoprotein D, GFP, and VP11/12 is reduced following treatment of HSV-1 with higher concentrations of BTE.
To determine if treatment with BTE interfered with the production of viral genomes, PCR was used to compare the relative levels of total DNA produced by infection with BTE-treated and untreated HSV-1. There was approximately a 75% reduction in the concentration of DNA in cells following treatment with 1.4 mM BTE (Table 3). Gel electrophoresis of the PCR products from DNA (extracted from HSV-1 infected A549 cells) resulted in visible bands on the gel corresponding to viral genes for glycoprotein D (gD), GFP and pUL46, apparent for untreated HSV-1 and HSV-1 treated with 1.4 mM BTE; however, the former had a higher intensity than the latter (Figure 5A). Sequence-specific primers were also used to amplify the viral DNA (extracted from HSV-1 infected Vero cells) encoding viral GFP at 12 hours post-infection for untreated HSV-1 or HSV-1 treated with varying concentrations of BTE (Figure 5B). The intensity of viral DNA products obtained after infection with untreated HSV-1 (column 2), was greater than that of HSV-1 treated with 0.14 μM, 1.4 μM, or 0.14 mM BTE (columns 3 – 5). Subsequent experiments focused on how higher concentrations of BTE affected HSV-1 infectivity.
Based on the NGS results, we designed HKU20-specific primers targeting a fragment of the N gene and re-screened the δ-CoV-positive samples for which we had sufficient amounts of RNA. Six out of 8 re-screened samples were positive in RT-PCR using HKU20-specific primers. Three samples yielded PCR fragments of sufficient quantity/quality and were used for capillary sequencing. Phylogenetic analysis of these fragments demonstrated that similar to blue-winged teal coronavirus/USA/Illinois2562/2017, the three δ-CoVs were most closely related to wigeon coronavirus HKU20 (Figure 3) sharing 83.38%–84.39% nucleotide identity in the N gene. The two samples positive in RT-PCR with UDCoV primers, but negative with HKU20 primers (Table 2) could contain insufficient amounts of δ-CoV RNA to generate a longer amplicon (targeted by the HKU20-specific primers) or could possess genetic characteristics distinct from HKU20 δ-CoV and other δ-CoVs identified in this study.
Thermotolerance is generally considered as a prime condition for vertebrate pathogenicity. During the last decades, several black yeasts have been described in Exophiala that constantly lacked thermotolerance, but still were associated with animal disease, indicating that these fungi have other, intrinsic, temperature-independent infectious abilities. Infections were especially within fish and amphibians, but sometimes also in invertebrates. Such infections seem to be relatively regular, at least in captive and farmed fish and amphibians. Outbreaks in farmed and aquarium animals could cause serious losses to aquaculture and fishery industries, but because of the spread nature of reports it is hard to estimate the magnitude of the problem.
Rolling circle amplification is a powerful and easy, isothermal in vitro DNA amplification method emerging as a tool for quick detection of specific nucleic-acid sequences in DNA samples. The use of a padlock probe to circularize oligonucleotides was produced by Nilsson. The technique is on the basis of the replication of a short, single-stranded DNA circle by Bst DNA polymerases at constant temperature. Sequencing of the internal transcribed spacer (ITS) is the gold standard for species recognition of black yeast and relatives, as it provides sufficient resolution between species. For analysis of large numbers of isolates in case of outbreaks and epidemiological monitoring, sequencing is nevertheless costly, time-consuming and impractical. Furthermore, validated databases for comparison are needed, as GenBank information is polluted with wrongly identified sequences; we used a research database on black fungi housed at the Westerdijk Fungal Biodiversity Institute and of which a selection has been deposited in the ISHAM ITS Database (www.its.mycologylab.org). The RCA reaction is relatively free of requirement for high priced laboratory equipment and could be done within 2 h isothermally at 65 °C in a water bath, thermocycler, heat block or microwave. Nevertheless, positive signals are often visible 15 min after commencement of the RCA reaction when recognized by real-time PCR [19, 22]. The amplification product can be visualized by agarose gel electrophoresis, but can also be visualized in gel-free methods applying fluorescence staining of amplified product by SYBR Green in combination with a UV transilluminator. The progress of RCA probes to distinguish single species or groups of species depends on the presence of adequate sequence information and useful species-specific polymorphisms in genes of precisely identified species.
The objective of the current study was to begin a screening technique based on RCA for highly specific and rapid detection of waterborne Exophiala species which repeatedly occur in the form of outbreaks in farmed fish, enabling their unambiguous differentiation from related melanized fungi. The RCA method performed well elsewhere in the fungal Kingdom, e.g., in Candida, Aspergillus, Scedosporium, Cryptococcus, Trichophyton, Fusarium, human-pathogenic Exophiala, Talaromyces marneffei, Scedosporium, Rhizopus and Fonsecaea. A low-cost alternative to RCA might be loop-mediated isothermal amplification (LAMP). This technique uses a set of six oligonucleotide primers with eight joining sites hybridizing particularly to various parts of a target gene. Najafzadeh et al. compared RCA and LAMP detection for human-pathogenic Fonsecaea species and discovered that LAMP was extremely sensitive, but RCA become more specific.
In conclusion, RCA is a very fast (less than 1 working day), specific (down to the single-nucleotide level) and economical (no additional equipment required) method for specific and rapid identification of fungal pathogens where large numbers of strains need to be processed. Our results show a considerable potential of the method in the future in laboratories for fungal outbreak control, e.g., in farmed fish. The establishment of the test is relatively expensive, but with high throughput applications, the final result per strain will be rapid and cost-effective.
Many hosts are members of multi-species aggregations and may be infected by an assemblage of specialist and/or multi-host generalist infectious agents. Host community diversity is central to microbial dynamics [1, 2], and species richness, relative abundance, specificity and intra- and inter-species interactions within assemblages likely have complex roles in modulating microbe levels within populations [1, 3–8]. A significant limitation in studying viral communities in hosts is that most viral species remained undescribed, such that viral ecology across multi-host systems has been limited to “single-virus” dynamics, particularly in vertebrate systems (for example, Influenza A virus [IAV] in avian populations). With the advent of unbiased, bulk ‘meta-transcriptomic’ RNA sequencing we can now explore, in more detail, how viral community structure may be shaped by host-species interactions.
Through long distance migration, wild birds connect the planet. Crucially, they are important reservoirs for viruses with negative consequences for wild birds (e.g. Wellfleet Bay virus;), poultry (e.g. Avian avulavirus 1;), and humans (e.g. IAV;). Despite their importance, we know little of avian viral communities. Birds of the orders Anseriformes and Charadriiformes, the central reservoirs for notifiable avian viruses such as IAV, avian avulavirus, and avian coronavirus [13, 14], form multi-host flocks, in which many species may migrate, forage, or roost together, making these groups excellent models for studying virome ecology. Flocks may comprise species along a taxonomically related gradient and may utilize similar or different ecological niches in the same environment. For example, in Australia, taxonomically related dabbling Grey Teals (Anas gracilis) and Pacific Black Ducks (Anas superciliosa) may share the environment with the distantly related filter feeding Pink-eared Duck (Malacorhynchus membranaceus). These multi-host flocks form multi-host maintenance communities, with consequences for virus ecology, transmission, and virulence [1, 16, 17].
Studies of the ecology of IAV, the best studied multi-host virus in wild birds, have shown that not all hosts are equal [18, 19]. In particular, there are marked differences in susceptibility, pathology and the subsequent immune response in related species, or more diverse species within similar ecological niches. For example, dramatic differences in viral prevalence exist within the Charadriiformes, such that Ruddy Turnstones (Arenaria interpres) may have an IAV prevalence of ~15%, compared to the negligible IAV prevalence in co-sampled Sanderlings (Calidris alba) at Delaware Bay, USA. There are also major differences in the pathology of highly pathogenic IAV in Anseriformes in field and experimental infections. Mallards (Anas platyrhynchos) infected with highly pathogenic IAV are thought to move the virus large distances and remain free of clinical signs, while Tufted Ducks (Aythya fuligula), in contrast, experience severe mortality [21–23]. Following IAV infection, dabbling ducks of the genus Anas are believed to develop poor immune memory, allowing life-long IAV re-infection, in contrast to swans that have long-term immune memory and where re-infection probability is likely very low in adults. These differences are driven by factors encompassing both virus (e.g. virulence, transmission) and host (e.g. receptor availability, immune responses) biology.
The goal of this study was to use the analysis of comparative virome structures, particularly virome composition, diversity and abundance, as a means to describe host-virus interactions beyond the “one-host, one-virus” model. Given their role as hosts for multi-host viruses, we used apparently healthy members of the Anseriformes and Charadriiformes as model avian taxa in these comparisons. In particular, using samples collected from Australian birds, we aimed to (i) reveal viromes and describe novel viruses in the bird taxa sampled, (ii) determine whether viromes of different host orders have different abundance and viral diversity, (iii) determine whether closely taxonomically related and co-occuring avian hosts have viromes that are more homogenous, and (iv) identify the impact of host taxonomy, which we use as a proxy for differences in relevant host traits (such as host physiology, cell receptors), in shaping virome structure. Overall, we reveal a combination of virome heterogeneity and connectivity across species that are important reservoirs of avian viruses.
Exophiala is a member of the ascomycete order Chaetothyriales (fungi), comprising the black yeasts and allies, which are frequently encountered as causative agents of disorders in humans and animals [1–7]. Human infections vary from commensalism or moderate cutaneous infection to fatal neurotropism with serious mutilation. Infections frequently involve patients without known immune disorder or underlying metabolic disease. Outside humans, especially cold-blooded waterborne vertebrates are susceptible to a variety of Exophiala species, some of these seem to be specific to certain host taxa. As virulence factors, the capability to absorb alkylbenzenes, within sweat and nervous tissues of mammals and in the poisonous skin of amphibians, has been proposed [2, 8, 9]. Studies on epizootics from the older literature obviously show that black yeast infection is a relatively popular phenomenon in cold-blooded vertebrates [10–15]. Recent molecular reports demonstrate that various pathogenic species are concerned [1, 2, 16], which morphologically are extremely similar.
Some species can be classified by physiological characteristics, for example, temperature tolerance and nitrate assimilation, however, for many taxa molecular characterization are needed.. Sequencing of the rRNA ITS location is generally adequate for routine species distinction in the genus Exophiala. This method is relatively costly and time-consuming and less suitable for large numbers of strains in case of monitoring of epizootics.
Rolling circle amplification (RCA) is an isothermal DNA amplification technique applying so-called padlock probes. The method has been proven to be fast, cost-effective and specific for identification of human and plant pathogenic fungi [6, 19–25], including black yeasts and relatives [26–28]. The 3′- and 5′-end strands of the probes hybridize next to one another at the target strand, leading to circularization of the molecule upon ligation. The circular molecule is consequently amplified isothermally with a DNA polymerase that lacks exonuclease activity, and the resulting product subsequently can be utilized with a second primer causing a cascade of amplifications. Because of the necessary accurate base pairing, the padlock probes have the ability to identify single position mutations [29–31].
In the present paper, we developed eight padlock probes on the basis of the ITS location to identify the most relevant species of Exophiala in animal infection and epizootics, viz. E. equina, E. salmonis, E. opportunistica, E. aquamarina, E. angulospora and E. castellanii, together with Veronaea botryosa as out-group. The objective of the current study was to evaluate the practical applicability of the method and to assess its limitations.
While γ-CoVs are endemic and highly prevalent in wild and domestic birds, δ-CoVs have only been discovered in the US only recently. Thus, the role of wild avian species in the ecology of δ-CoVs in the US is unknown. Environmental sampling of high-risk and co-mingling sites in Alberta and Saskatchewan Canada, identified δ-CoV in sparrows, that are closer phylogenetically to porcine δ-CoVs than to those from waterfowl. To date, the presence of δ-CoV is confirmed in sparrows in Canada and the US, in quail in Brazil and in various birds in Australia, suggesting that δ-CoVs circulate in different avian species in the Americas. Since porcine δ-CoV often results in severe clinical disease and mortality in piglets, which impacts the swine industry, it is important to understand the morbidity and interspecies transmission rates between birds and pigs. Due to their flocking behavior and abilities to fly long distances, birds can play a role in the dissemination of δ-CoVs among themselves and other animals.
The apparent prevalence of δ-CoV in our study was only 1.13%. However, this is higher compared with that observed in our previous study (0.5%) that analyzed Ohio samples from the period 2013–2014. Also, of these 14 positive samples, only up to 4 originated from the same state, suggesting that δ-CoVs spread with low efficiency in the avian species tested. Additionally, our inability to recover CoV sequences by NGS suggests that they are present in the samples at lower frequency compared with other microorganisms. The latter is more suggestive of asymptomatic carrier status as opposed to acute infection associated with clinical disease. These findings suggest that avian species likely represent a natural reservoir of δ-CoVs, while pigs and other mammals serve as spillover hosts.
The higher prevalence of γ-CoVs in the US of 4.99% is consistent with findings from some previous studies. In a screening study from Brazil, quail were susceptible to both δ- and γ-CoV. The higher prevalence of γ-versus-δ-CoVs and variations in preferred avian host species have been also reported by other researchers in other countries, such as Australia, Asia (Hong Kong and Cambodia) and Finland. This may indicate δ-CoV emerging status and its ongoing spread in the US.
In this study, a higher prevalence of δ- and γ-CoVs in aquatic vs. terrestrial birds was evident, with δ- and γ-CoVs prevalence in aquatic birds being 1.34% and 6.3%, respectively, compared with only 0.6% and 0% in terrestrial birds. This suggests that aquatic birds may represent a natural reservoir for CoVs of terrestrial birds and pigs, and their concentration or survival may be increased in water sources compared with other avian habitats. Similarly, in a surveillance study in Hong Kong and Cambodia, δ-CoV was found in different wild aquatic bird species including gray herons, pond herons, great cormorants, black-faced spoonbills, and several duck species, whereas γ-CoVs were found in little whistling ducks, tufted ducks, common teals, northern shovelers, eurasian wigeons, and northern pintails. In wild Australian birds, δ-CoV was detected in pacific black ducks, but it was also detected in terrestrial birds such as curlew sandpiper, red-necked stints, ruddy turnstones, and pied herons. This suggests that wild birds are major reservoirs of a wide range of δ- and γ-CoVs, and the circulation of CoVs without association with clinical disease is more common than previously recognized. However, it is important to note that δ-CoVs identified in terrestrial birds and pigs are more similar each other phylogenetically, and those from aquatic/wading birds are genetically more distinct. It is of interest, that prevalence of both γ- (92%) and δ-CoVs (86%, Table 1) was higher in colder (October–February) than in warmer (March–September) months. This indicates that, similar to previous findings on avian, animal and human CoVs, there are may be seasonal (and migration associated) fluctuations in the prevalence of avian coronaviruses in wild bird in the US.
Because phylogenetic analysis of the partial N gene of the 4 newly identified δ-CoVs (Table 2) revealed that they were most closely related to HKU20 (wigeon) δ-CoV, we hypothesized that it may be the parental strain recently introduced into the US.
In our study, these newly identified δ-CoVs were more closely related to other δ-CoV from aquatic birds, but not terrestrial avian species and pigs, which can be explained by the fact that aquatic birds occupy a separate ecological niche, while terrestrial birds and pigs may share some co-mingle sites. Although, we did not identify δ-CoV strains genetically similar to sparrow and porcine δ-CoVs, it is important to note that porcine δ-CoV outbreaks in the US were predominantly detected in the states with the highest density of pig population that in turn have a significant geographical overlap with the Mississippi Flyway (Figure 4). While a small pilot study failed to identify porcine δ-CoVs in wild waterfowl in the Mississippi Flyway, the overlap of the bird migratory pathways and high density swine farms creates favorable conditions for birds and pigs to exchange δ-CoV pools directly or through some other intermediate hosts.
We examined the relative basal expression levels of TLR3 and TLR7 mRNA in naive PBMC by quantitative real-time RT-PCR (qRT-PCR) in nine individual animals per breed (n = 9). The Kanni and Salem Black breeds revealed significantly higher basal TLR3 (p<0.001) and TLR7 (p<0.001) transcripts than Barbari goats while there were no significant difference (p>0.05) between Tellicherry and Barbari breeds (Figure 1). To understand their contribution to virus replication, PBMC from each of these four goat breeds (n = 5) were infected with 1×103.0 mean tissue culture infective dose (TCID50) of PPRV and the virus load analyzed at 24 h post infection (PI) by qRT-PCR, using primers specific to the PPRV-H gene and TCID50. PBMC from Barbari and Tellicherry goats supported significantly (p<0.01) higher PPRV replication than those from Kanni and Salem Black (Figure 2A) with the yields being similar in these two breeds. There was a significant reduction in virus yield by one log10 in Kanni/Salem Black PBMC (p<0.05) compared to Barbari/Tellicherry goats (Figure 2A).
We then examined if pre-stimulation of PBMC with the TLR agonists poly I:C and imiquimod would result in reduction in PPRV replication. A difference of about 6–8 Ct values in PPRV-H gene mRNA levels was observed between unstimulated and poly I:C treated PBMC (Figure 2B). In the case of pre-stimulation with imiquimod, this difference was about 2–3 Ct values for Barbari and Tellicherry breeds and about 4–5 Ct values for Kanni and Salem black breeds (Figure 2C). Comparison of viral load (PPRV-H gene mRNA levels and infective viral titres) in all the breeds following imiquimod treatment indicates a significant (p<0.05) reduction of viral load in Kanni and Salem black breeds than Barbari and Tellicherry breeds. TCID50 was not determined for poly I:C treated and PPRV infected PBMC since the 40-Ct values of PPRV H mRNA expression levels across these breeds were not significantly different (Figure 2B).
The complete and validated sequence assembled using reads from snake no. 2 has been deposited with the NCBI under accession no. KJ541759.
In this report, we describe a severe respiratory disease affecting ball pythons, and we have identified a nidovirus as a candidate etiologic agent by means of association. We collected cases from around the United States and performed a variety of pathological and diagnostic analyses. Respiratory tract pathology was consistent with a viral etiology, as was the presence of virus-like particles in lung epithelium of affected snakes. Using unbiased deep sequencing, we identified and assembled the genome of a previously uncharacterized nidovirus. This virus shares several attributes with other nidoviruses but has several distinguishing characteristics, including its record-setting genome size and reptile host.
The findings presented here raise a number of questions related to the biology of this virus and its relationship to disease. Foremost among these is whether this virus causes the observed respiratory pathology. Several observations suggest a causal relationship. First, detection of viral RNA correlates with clinical signs. Second, the virus load is most profound in the respiratory tract, the site of disease. Third, other animal nidoviruses cause severe respiratory disease. Nevertheless, formal evidence of disease causality remains to be demonstrated. Although virus isolation attempts have yet to succeed, experimental infections using material prepared directly from infected tissues could fulfill Koch’s postulates.
The evolutionary relationship between ball python nidovirus and other nidoviruses is partially clear. In analyses based on the highly conserved replicase subunits, this virus clearly clusters with viruses in the subfamily Torovirinae, the toroviruses found in mammals and the bafiniviruses found in ray-finned fish, with 100% Bayesian posterior probability and ML bootstrap support (Fig. 7; see also Fig. S6 in the supplemental material). This suggests that at least for the core replicase gene, the snake virus shares a common evolutionary origin with these mammal and fish viruses. Although it has not been proven that the virus sequence corresponds to the particles observed in electron micrographs, the ultrastructural appearance and size of the observed virus are notably similar to those of virions of white bream virus in the genus Bafinivirus (61, 62). We propose that ball python nidovirus establish a new genus in the Torovirinae named Barnivirus, for bacilliform reptile nidovirus, a naming convention borrowed from the genus Bafinivirus.
While some studies have found support for the monophyly of Torovirinae and Coronavirinae, in accordance with the current International Committee on Taxonomy of Viruses (ICTV) status of Coronaviridae, our analysis did not find phylogenetic support for this grouping, in agreement with other recent analyses (1, 40, 42, 63). The paraphyly of Coronaviridae was supported by log likelihood testing. The availability of a more complete representation of existing species for comparison results in greater phylogenetic resolution (64). Significant errors can occur in phylogenetic analyses due to incomplete taxum sampling, even if very large sequence length is assessed (65). This further emphasizes the need for exploration of viral diversity and increased sampling of the Nidovirales.
Current standards in herpetoculture encourage disease transmission and may foster evolution of increased pathogen virulence. Captive snake breeding operations typically operate at high stocking densities, and breeders commonly attend trade shows, where animals from different sources are juxtaposed. In addition, animals from geographically and ecologically diverse areas are commonly imported and mixed with minimal quarantine. These practices increase pathogen exposure and lower barriers to transmission. The outcome is evident in the case of farmed turtles, which typically have very high Salmonella carriage rates, in contrast to the low prevalence in wild turtle populations (66–68). It is also possible that these practices select for increased virulence, as has been the case for feline calicivirus (FCV), which has independently evolved multiple times from a pathogen that causes a relatively benign upper respiratory disease to one that causes hemorrhagic disease with high mortality (69). This increase in FCV virulence has been documented only in high-density environments, such as shelters. Surveillance of wild ball pythons in Africa will shed light on whether a similar phenomenon has occurred here. Further investigation of the pathogens of reptiles and improved biosecurity practices and disease surveillance of the herpetoculture industry are indicated.
In addition to disease causality, a number of questions related to the biology of ball python nidovirus and to the epidemiology and natural history of this disease remain open. The precise host range of this virus remains undefined. It is not clear that ball pythons are the primary natural host for this virus, nor whether it can replicate or cause disease in other snakes, other reptiles, or other animals. The routes of transmission for this virus remain unknown. Additionally, the prevalence of this disease is yet to be determined, although it is apparently widespread, since cases were collected from geographically disparate locations in the United States from 2006 to 2013. Additional studies will help to answer these central questions and uncover additional details about the biology of this putative pathogen. The current study is another example where the application of genomic techniques to the study of infectious disease in reptiles and other less well studied organisms has identified new and unexpected relatives of human pathogens (59, 70).
The interspecific relationships of Rattus using the mitogenome and cyt b sequences (n=396) were well-resolved (BS=95–100) or moderately resolved (BS=55–77) (Figure 5). Sequences representing animals from China fell into seven lineages that corresponded with R. nitidus, R. norvegicus, R. exulans, R. andamanensis, R. losea, R. rattus, and R. tanezumi. The clade of R. nitidus had two subclades, one from southern Xizang and the other from southeastern China (Figure 5). The tree depicted GenBank sequences deposited under different names within a shallow clade, most commonly with R. andamanensis. However, some specimens were also associated with R. losea, R. nitidus, R. tanezumi as well as R. nitidus from southern Xizang.
Here, we attempt to investigate MERS-CoV demographic patterns in the camel reservoir. We supplement camel sequence data with a single earliest sequence from each human cluster, treating viral diversity present in humans as a sentinel sample of MERS-CoV diversity circulating in camels. This removes conflicting demographic signals sampled during human outbreaks, where densely sampled closely related sequences from humans could be misconstrued as evidence of demographic crash in the viral population.
Despite lack of convergence, neither of the two demographic reconstructions show evidence of fluctuations in the scaled effective population size (Neτ) of MERS-CoV over time (Figure 5). We recover a similar demographic trajectory when estimating Neτ over time with a skygrid tree prior across the genome split into ten fragments with independent phylogenetic trees to account for confounding effects of recombination (Figure 5—figure supplement 1). However, we do note that coalescence rate estimates are high relative to the sampling time period, with a mean estimate of Neτ at 3.49 years (95% HPD: 2.71–4.38), and consequently MERS-CoV phylogeny resembles a ladder, as often seen in human influenza A virus phylogenies (Bedford et al., 2011).
This empirically estimated effectived population can be compared to the expected effective population size in a simple epidemiological model. At endemic equilibrium, we expect scaled effective population size Neτ to follow I/ 2β, where β is the equilibrium rate of transmission and I is the equilibrium number of infecteds (Frost and Volz, 2010). We assume that β is constant and is equal to the rate of recovery. Given a 20 day duration of infection in camels (Adney et al., 2014), we arrive at β=365/20=18.25 infections per year. Given extremely high seroprevalence estimates within camels in Saudi Arabia (Müller et al., 2014; Corman et al., 2014; Chu et al., 2014; Reusken et al., 2013; Reusken et al., 2014), we expect camels to usually be infected within their first year of life. Therefore, we can estimate the rough number of camel infections per year as the number of calves produced each year. We find there are 830,000 camels in Saudi Arabia (Abdallah and Faye, 2013) and that female camels in Saudi Arabia have an average fecundity of 45% (Abdallah and Faye, 2013). Thus, we expect 830 000×0.50×0.45=186 750 new calves produced yearly and correspondingly 186,750 new infections every year, which spread over 20 day intervals gives an average prevalence of I=10 233 infections. This results in an expected scaled effective population size Neτ=280.4 years.
Comparing expected Neτ to empirical Neτ we arrive at a ratio of 80.3 (64.0–103.5). This is less than the equivalent ratio for human measles virus (Bedford et al., 2011) and is in line with the expectation from neutral evolutionary dynamics plus some degree of transmission heterogeneity (Volz et al., 2013) and seasonal troughs in prevalence. Thus, we believe that the ladder-like appearance of the MERS-CoV tree can likely be explained by entirely demographic factors.
Since Bid plays important role in virus-induced apoptosis, we tested whether Bid-deficient rare minnow could modulate the GCRV-induced death compared with the wild type rare minnow. As shown in Figure 6, following infection with GCRV, the wild-type rare minnow started to die at as early as the fourth day, and all of them died in 6 dpi with a median survival time of 5 days. In contrast, Bid-deficient rare minnow started to die at the fifth day, which continued to the ninth day post GCRV stimulation (Figure 6). Moreover, the median survival time of Bid-deficient rare minnow after infected with GCRV (6 days) was more than that of wild-type rare minnow (5 days). Obviously, the results revealed that Bid-deficient rare minnow delayed the death induced by GCRV infection.
Mycetoma is a unique tropical disease, endemic in many tropical and subtropical regions that has been recently added to the WHO list of neglected tropical diseases.. It is mainly prevalent in what is known as “mycetoma belt” which includes Mexico, Senegal, Sudan, India and other countries between tropic of cancer. In 2014, a mycetoma consortium of scientists and physicians published research gaps on mycetoma which need to be addressed in the coming years. One of the research priorities identified was the need to develop a reliable and cost-effective method for species identification to improve diagnosis.
Mycetoma agents have been extensively studied in recent years,,. The large phylogenetic distance between a number of these agents provides the possibility to use a moderately variable marker like rDNA ITS for species identity. Ahmed et al. developed PCR-restriction fragment length polymorphism (RFLP) for identification of M. mycetomatis targeting the ITS region. However, with the description of the molecular siblings M. fahalii, M. pseudomycetomatis, and M. tropicana
 the method might be insufficiently accurate. Moreover, there is a need for identification these siblings species; Madurella grisea appeared to be distantly related and was re-named as T. grisea
In the present study we developed a simple, fast and highly specific molecular method for the identification of agents of black grain mycetoma. In this method, the ITS region is easily amplified using one set of primers, which simplifies the use. In a second, isothermal amplification reaction padlock probes are used to identify the species by RCA. The only equipment necessary is a thermocycler for the PCR reaction and a water bath or heating block for the RCA reaction. This relative simplicity enhances possible use in routine laboratories in endemic areas. Due to its robustness, high potential, and reproducibility, RCA is increasingly used as a diagnostic tool in pathogenic fungi, e.g. agents of chromoblastomycosis, dermatophytes, Aspergillus, Candida, and Talaromyces marneffei
,,,. The method does not require DNA sequencing and is therefore considered as a rapid and cost-effective. Applications are being expanded to nano- and biotechnology.
In the present study eight species-specific probes were designed and used for identification of 62 isolates. For the RCA reaction species probe hybridization to the 3′ and 5′ ends of target DNA and joining of adjacent ends by DNA ligase when both show perfect complementarity. The ligation appears to be highly specific and thus the method can detect single nucleotide polymorphism. The amplification reaction is driven by an isothermal DNA polymerase to amplify the circularized probes with high efficiency and an estimated capacity to synsthesize more than 70,000 bp per hour. RCA products can be detected with different methods including gel electrophoresis, radiolabeling, UV absorbance, fluorescence, and single molecule detection. It was known that the positive signals can be detected within 15 min after starting the RCA reaction by real time PCR. In the present study the RCA positive signal was easily visualized using both gel electrophoresis and fluorescent dye. The duration of our RCA protocol was 2 h, but additional time is required for DNA extraction and ITS amplification. Compared to the DNA sequencing the turnaround time for RCA is 2 hours less than sequencing and this even more if there is no in-house sequencer available.
Our results with eight padlock probes showed that RCA accurately identified all species with no cross reactivity (Fig. 1). It may be concluded that RCA is extremely useful for specific identification of agents of mycetoma. Performance and rapid turnaround time features make the RCA suitable for quick and reliable diagnosis, which is an enormous improvement compared to the current phenotypic identification of mostly non-sporulating cultures. Future application of RCA could be the detection of agents DNA directly from clinical samples without requirement of culturing.