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Severe fever with thrombocytopenia is a newly recognized disease in rural areas of northeastern and central China, with several cases in Japan and South Korea (Promedmail, 2013). Caused by SFTSV, the transmission route of the virus is still unknown, but most likely involves arthropod vectors or animal hosts since the virus has been detected in ticks collected from domestic animals (Tian et al., 2017), and the animals (i.e., goats, cattle and dogs) also have high levels of SFTSV-specific antibodies (Jiao et al., 2012). Patients infected with SFTSV present with fever, vomiting, diarrhea, thrombocytopenia, leucopenia, and increased liver enzyme levels, in which severe cases of SFTSV eventually result in multiple organ failure resulting in death (Yu et al., 2011). The fatality rate amongst hospitalized patients can be up to 30%, and hundreds of cases are reported annually in China (Liu et al., 2015).
Infection of wild-type mice (BALB/c, C57BL/6) results in limited weight loss but the animals do not succumb to disease (Chen et al., 2012; Jin et al., 2012). In one study, Liu et al. (2014) infected 6–10 week old Ifnar–/– mice (129/Sv background) SC with 106 ffu of SFTSV strain YL-1. The mice were highly susceptible to challenge, with all mice appearing ill by 3 dpi, resulting in death between 3–4 dpi. Blood and major organs (brain, heart, kidney, intestine, liver, lung and spleen) were collected from infected Ifnar–/– mice daily, and results showed high levels of virus replication with systemic spread to all organs. In particular, the spleen and intestine had the highest peak virus titers at death (Liu et al., 2014). In another study, Matsuno et al. infected 6–12 week old Ifnar–/– mice (C57BL/6 background) with either a high dose (105 TCID50 per animal) or a low dose (102 TCID50 per animal) of SFTSV strain SD4 via the intradermal (ID), IP, IM or SC routes. The results showed that the Ifnar–/– mice were susceptible to infection via all routes, with animals succumbing to death at 4 and 6 dpi in the high and low dose groups, respectively (Matsuno et al., 2017).
HeV was discovered in 1994 as the etiologic agent that caused an acute respiratory disease in horses in Australia with sporadic but lethal transmission to humans, with one fatal case developing pneumonitis, respiratory and renal failure, arterial thrombosis, and eventually cardiac arrest seven days after admission (Selvey et al., 1995). HeV currently still poses a threat to Australian livestock, and the CFR is estimated to be 60% for humans and 75% for horses (Field et al., 2011). NiV was discovered in 1999 in Malaysia with spread to neighbouring Singapore, resulting in 100 deaths from 257 human cases (CDC, 1999a). Patients typically present with respiratory problems and fever, as well as encephalitis with symptoms of headache, drowsiness, disorientation and confusion, rapidly progressing to coma. Since then, outbreaks of NiV have caused severe encephalitis in Bangladesh and India, with a CFR of ~75% (Lo & Rota, 2008). Pigs are susceptible to infection and act as amplifying hosts to humans (CDC, 1999b). Fruit bats are the natural reservoir for both viruses (Halpin et al., 2011).
Wild-type mice are only susceptible to HeV or NiV infection if the virus is administered via the intracranial (IC) route, but not through any other types of inoculations (Dhondt et al., 2013). In their study, Dhondt et al. infected 3–18 week old Ifnar–/– mice (C57BL/6 background) IP with 106 pfu of HeV. It was observed that while the infection was fully lethal in 3-week old mice, the susceptibility decreased with increasing age and the same dose of HeV in 18-week old mice only resulted in 50% mortality. The moribund mice died between 7–13 dpi. For NiV strain UMMC1, 4–12 week old Ifnar–/– mice (C57BL/6 background) were infected IP with increasing dosages from 100–106 pfu. The mice were found to be uniformly susceptible with deaths between 6–9 dpi in the 106 pfu group, and the LD50 was calculated to be 8×103 pfu in Ifnar–/– mice. Infected mice with both viruses first showed behavioural changes including agitation, edginess and no grooming. Neurological symptoms were observed with advanced disease including tilted head and paralysis. A weight loss of approximately 15%–25% was observed 1–2 days before death and found to be a good predictor of mortality (Dhondt et al., 2013).
PCRs testing were repeated on the 50 fruit bats original samples including the Kidney, heart, lung, liver, spleen, intestine, rectal swab sample, and brain samples. Two bat’s QPCRs results were positive. One bat’s QPCRs result was positive in the lung, intestine sample (Cangyuan virus isolated) and rectal swab sample, and the Ct (Threshold Cycle) of QPCR were 19.86 ± 0.056, 19.52 ± 0.041, 19.64 ± 0.061 respectively. The Ct of another bat’s PCR were 23.07 ± 0.253, 22.53 ± 0.171 in the intestine sample and rectal swab sample, respectively.
To establish the evolutionary relationship between Cangyuan virus and other known orthoreoviruses, Homology were compared (Table 2, Table 3 and Additional file 1: Table S1, Additional file 2: Table S2 and Additional file 3: Table S3) and phylogenetic trees were constructed based on the nucleotide sequences of the L genome segments (Figure 2), the M genome segments (Figure 3) and the S genome segments (Figure 4). The Cangyuan virus L1-L3, M1-M3 segments sequence identity were 81.6% –94.2%, 83.8%–97.9%, 85.9%–97.6% ( Additional file 1: Table S1), 82.2%–94.1%, 78. 1%–95.0%, and 83.0%–93.9% (Table 2, Additional file 2: Table 2), respectively, by alignment with Pteropine orthoreovirus (PRV) species group. The phylogenetic trees for L2, L3, M1 and M2 segments demonstrated that Cangyuan virus was most closely related to Melaka and Kampar viruses, and was placed in Pteropine orthoreovirus (PRV) species group which covers all known bat-borne orthoreoviruses together with Nelson Bay orthoreovirus.
To better understand the genetic relatedness of Cangyuan virus to other known bat-borne orthoreoviruses, the published sequences for the S genome segment of bat-borne orthoreoviruses known for causing acute respiratory disease in humans were retrieved from GenBank and used to compare homology (Table 3 and Additional file 2: Table S2) and construct phylogenetic trees (Figure 4). The Cangyuan virus S1-S4 segments sequence identity were 55.3%–94.7%, 86.2%–95.5%, 86.5%–97.9%%, and 83.5%–98.2%, respectively (Table 3 and Additional file 2: Table S2). The S1 segment demonstrated a greater heterogeneity than other S segments in Pteropine orthoreovirus (PRV) species group.
The MERS-CoV agent is a betacoronavirus first described in September 2012 from a patient in Saudi Arabia. It is related to the severe acute respiratory syndrome coronavirus (SARS) that from 2002 to 2003 resulted in over 8000 infections in 17 countries (including Macau and Hong Kong) with a 10% fatality rate. In the case of SARS, bats were confirmed as the natural reservoir although the introduction of the agent to humans was likely through contact with animals (masked palm civets) held in southern Chinese markets where the disease was first recognized in Nov 2002. Middle East respiratory syndrome coronavirus also likely represents a zoonotic agent that infected humans via viral adaptions or a species jump through an intermediate host. Bats are suspected as the ultimate reservoir with camel infection as the most likely link to human infection, but these theories at present are unproven. The majority of cases are associated with severe disease of the lower respiratory tract resulting in pneumonia and multi-organ failure; the most susceptible to clinical disease and death are those with pre-existing co-morbidities. Other chronic conditions have been reported in 96% of clinical cases including diabetes, hypertension, heart disease and kidney disease. It is unclear whether persons with these specific conditions are disproportionately infected or have more severe disease. To 30 November 2013, as reported by the US Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO), ten countries have reported 160 clinical cases with 68 deaths. All cases and transmissions have been associated with the Arabian Peninsula where the vast majority of cases and deaths have occurred; travel of infected patients with limited person-to-person transmission are responsible for cases reported elsewhere. Although it appears that there is limited person-to-person transmission, clusters of cases have been described. A cluster of 21 human cases occurred associated with four hospitals in Eastern Saudi Arabia that was ultimately sourced to one community-acquired case. Updated MERS-CoV rapid risk assessments are routinely published by the European Centres for Disease Control (ECDC). Regarding actions with respect to blood safety, careful surveillance is required as risk involving MERS-CoV is rapidly evolving at present. To date, only one study has reported testing blood donors including 110 in Saudi Arabia; all were nonreactive for MERS-CoV neutralizing antibodies. There is no evidence of transfusion transmission.
Venezuelan equine encephalitis (VEE) is an arbovirus infection transmitted by mosquitoes. VEE viruses (VEEV) are classified in the genus Alphavirus, family Togaviridae. The VEE virus complex is composed of six subtypes (I–VI); Subtype I includes five antigenic variants (AB–F), of which variants 1-AB and 1-C are associated with epizootics in equines and concurrent epidemics in humans. The epizootic variants 1-AB and 1-C are thought to originate from mutations of the enzootic 1-D serotype. The enzootic strains are 1-D, 1-E and 1-F of subtype I, subtype II, four antigenic variants (A–D) of subtype III, and subtypes IV–VI. The enzootic viruses do not produce clinical encephalomyelitis in the equines normally. Enzootic VEE strains have been identified as Everglades (subtype II) in the Florida, variant I-E in Central American countries and Mexico, variants I-D and I-E in Panama, variant I-D in Venezuela, Colombia, variants 1-D, III-C, and III-D in Peru, variant III-B and subtype V in French Guiana, variant I-D in Ecuador, variant III-A in Suriname and Trinidad, variants I-F and III-A and subtype IV in Brazil and subtype VI in Argentina. In an atypical ecological niche, variant III-B has been isolated in the USA (Colorado and South Dakota) in an unusual association with birds. Countries with incidence/serological evidence are presented in Fig. (4).
The primary vectors for the bird or rodent-mosquito life cycle are members of the Melanoconion subgenus (Culex cedecci). Epizootic VEEV strains (I-AB and I- C) are transmitted by several mosquito vectors (e.g., Aedes and Psorophora spp.) to equids.
Infections with VEE virus (VEEV) may present, in both humans and horses, as either encephalitic disease or as simply a febrile disease without profound neurologic signs. Horses may die after a very acute course, even without any neurologic signs, but mortality in humans is generally low. Horses are not dead-end hosts for VEEV epizootic strains like they are for EEEV and WEEV. Horses are, in fact, the key reservoir species for the epizootic strains of VEEV that cause clinical disease in both horses and humans.
Epizootic subtypes highly pathogenic to equines, can spread rapidly through large populations. Equines are the primary animal species and serve as amplifying hosts for epizootic VEE virus strains. Blood-sucking insects feed on infected horses, pick up this virus and transmit to other animals or humans. Animal like cattle, swine, and dogs, can become infected, but they neither show the signs of the disease nor contribute to spread. Aerosol transmission has been reported in human from laboratory accidents [128, 132]. Infections with both epizootic and enzootic variants are infectious to human beings and can occur in laboratory workers. The workers handling infectious VEE viruses or their antigens should take preventive measures including use of containment facilities and vaccination.
VEE can cause disease in equines including horses, mules, donkeys and zebras. Cattle, swine, chickens and dogs have been shown to seroconvert after epizootics; mortality has been observed in domesticated rabbits, dogs, goats and sheep. Humans also can contract this disease. Epidemics of VEE involving tens of thousands of humans have been reported. The mortality rates in equines during epizootics have been 19-83% while 4-14% in human beings associated with neurological disease
It usually causes influenza like symptoms in adults, but in children and horses it can cause severe encephalitis. Equines may suddenly die or exhibit progressive central nervous system disorders. Infections with VEEV may present, in both humans and horses, as either encephalitic disease or as simply a febrile disease without profound neurologic signs. Horses may die after a very acute course, even without any neurologic signs, but mortality in humans is generally low. Young and immune compromised horses are most likely to develop clinical signs. It causes only low morbidity and mortality in man but high morbidity and mortality in animals.
The epizootic VEE was initially limited to northern and western South America in Venezuela, Colombia, Ecuador, Peru and Trinidad, but the epizootics have been reported in years from 1969 to 1972 in Guatemala, Nicaragua, El Salvador, Honduras, Costa Rica, Belize, Mexico, and the United States of America due to variant 1-AB. Epizootics caused by I-AB or I-C virus have not occurred in North America and Mexico after 1972. Equine and human epizootic VEE viruses were subtype 1-C from Venezuela in 1993, 1995 and 1996 and Colombia in 1995. In 1960 over 200,000 human cases and more than 100,000 equine deaths were estimated in Central Colombia. Countless cases in horses and 75,000-100,000 human cases with more than 300 fatal encephalitis cases occurred in Venezuela and Colombia in 1995. Equine disease associated with VEEV-IE occurred in Mexico and human cases of VEEV ID-associated disease occurred in Peru from 1993 to 95. Subtype II has been isolated from humans and mosquitoes from Florida; subtype III has been isolated from the Rocky Mountains and northern plains states. Sylvatic VEE viruses are endemic in North, Central, and South America in swampy environments with persistent fresh or brackish water. Epizootics have been associated with a mutation to a subtype I (A, B, C, and possibly E), a change in mammalian pathogenesis, and change to several bridge vectors.
Treatment of viral encephalitis is supportive, as there are no specific antiviral therapies. The two VEE vaccines, a modified-live vaccine (TC-83) and an inactivated adjuvant vaccine, have been used in field. Horses were vaccinated with TC-83 vaccine during outbreak in Mexico and Texas in 1971 as equine vaccine was not available but it is still in use for humans working with VEE. Formalin-inactivated virulent VEE virus vaccines are not recommended for use in equids due to risk of residual virulence.
Western equine encephalitis (WEE) is an uncommon viral illness of horses and human. WEE virus (WEEV) is an Alphavirus of the family Togaviridae which is maintained between birds and mosquitoes, occasionally causing disease in humans and equids [135, 136]. This is an arbovirus transmitted by mosquitoes of the genera Culex and Culiseta. It is a recombinant between Sindbis and Eastern equine encephalitis like viruses. It has also been reported to cause disease in poultry, game birds and ratites. WEEV is normally maintained between Culex tarsalis mosquitoes and birds. WEE has several subtypes consisting Sindbis, Aura, Ft. Morgan and Y 62–33. WEEV previously isolated in the south and eastern USA has been shown to belong to the HJ virus serogroup.
Horses and humans are often referred to as “dead-end” hosts as the virus does not build to high enough levels in blood to infect other mosquitoes. Most people infected with WEE virus will have either no symptoms or a very mild illness. A small percentage of people, especially infants and elderly people to a lesser extent, may develop encephalitis. Approximately 5-15% of these encephalitis cases are fatal, and about 50% of surviving infants will have permanent brain damage.
Geographically, WEEV exists throughout uine deaths were estimated in central America and northern portions of South America, Mexico and Canada. In the US, WEEV exist in the western two third of the country. Outbreaks of the disease have been recorded since 1847. In 1930 about 6000 horses and mules were infected leading to about 50% mortalities in California. The largest epidemic was recorded in 1937 and 1938 in USA and Canada. In 1938 outbreak an estimated 264000 equids were infected with a morbidity of 21.4%. In the USA, WEE is seen primarily in provinces west of the Mississippi River. During 1941, there was an outbreak of WEE in several states of US and Canada causing 300,000 cases of encephalitis in mules and horses and 3336 cases in humans. The 1970s saw 209 human cases; 87 were reported during the 1980s, only 4 cases during the 1990s, and no cases have been reported in the USA or Canada since 1998. The last documented human case in North America occurred in 1994, and the virus has not been detected in mosquito pools since 2008. In human, WEEV infections tend to be asymptomatic or cause mild disease after a short incubation period of 2–7 days with nonspecific symptoms, e.g., sudden onset of fever, headache, nausea, vomiting, anorexia and malaise. In some cases, additional symptoms of altered mental status, weakness and signs of meningeal irritation may be observed. In a minority of infected individuals, encephalitis or encephalomyelitis occurs and may lead to neck stiffness, confusion, tonic-clonic seizures, somnolence, coma and death. WEEV is considered as agent that the US researched as potential biological weapons before the nation suspended its biological weapons program.
In horses, infections with WEEV begin with fever, inappetence and lethargy, progressing to various degrees of excitability and then drowsiness, ultimately leading to paresis, seizures and coma in 5-10 day course of the disease. The WEEV mortality rate in horses is higher than humans. Mortality of horses showing clinical signs of WEE is 20–50%. These symptomatic horses either progress to recumbency or die from WEE infections.
There is no treatment for WEE other than supportive care. Formalin-inactivated whole viral vaccines for EEE, WEE, and VEE are commercially available in mono-, bi-, or trivalent form. Previously non vaccinated adult horses require booster. For adult horses in temperate climates, an annual vaccine within 4 wk of the start of the arbovirus season is recommended. However, for horses that travel between areas affected by the virus, 2 or even 3 times vaccination in a year is recommended. Mares should be vaccinated 3–4 wk before foaling to induce colostral antibody.
A larger number of AstVs were detected in both rodent and shrew samples (Additional file 1: Table S4). Fifty-five AstVs were selected for sequencing. Most of the rodent AstVs sequenced belonged to four main genetic lineages 1 to 4 within the genus Mamastrovirus and had less sequence similarity with AstVs in other hosts (Fig. 5c). One rodent AstV, RtRn-AstV-1/GD2015, was closely related to AstVs of cattle, deer, and pigs with > 90% nt identity. Two shrew AstVs, Shrew-AstV/SAX2015 and Shrew-AstV/GX2016, were related to mouse AstV with ~ 70% nt identity in the genus Mamastrovirus. Lineage 5 contained one shrew AstV and one mouse AstV, with 79% nt identity with each other. Lineage 5 branched out of the genus Mamastrovirus and showed a closer relationship with the genus Avastrovius.
Dengue viruses include four genetically related viruses (65% genetic homology between the four) classified as ‘arboviruses’ (arthropod-borne viruses). All arboviruses involve replication through an arthropod host (usually a mosquito or tick) have a worldwide distribution and are responsible for huge periodic epidemics. Dengue viruses are in the family Flaviviridae along with related viruses: West Nile virus, yellow fever virus and over 65 other related viruses. Arboviruses also include hundreds of agents, of which many are human pathogens in other viral families (Bunyaviridae and Togaviridae including the alphavirus, chikungunya described below). Dengue viruses are the most important of all of these agents due to the number of human infections, clinical disease, associated deaths and ongoing expansion worldwide. It is estimated by the WHO that greater than 100 countries are endemic in the tropics and subtropics with over 2·5 billion people at risk. It is impossible to know the number of clinical cases that actually occur each year, but one estimate of the global burden of dengue during the 2010 worldwide pandemic was 390 million infections across four continents of which 96 million were symptomatic including 500 000 cases of severe dengue. In Asia and Latin America, dengue is the leading cause of hospitalization in children. Since there is no effective vaccine or specific treatment, vector control is the only intervention. The dengue cycle involves humans and mosquitoes (primarily A. aegypti), but sylvatic cycles also occur and are recently being investigated associated with preliminary reports of a new dengue virus type (DENV-5). Immunity to a given viral type is considered lifelong, but due to incomplete antibody neutralization between types, secondary infection with a different type can lead to more severe disease referred to ‘severe dengue’ including haemorrhagic fever and/or shock. The majority of clinical cases are classified as ‘fever’ defined by WHO as fever plus two other symptoms most commonly including chills, painful eyes, body aches (that can be severe, ‘break-bone fever’), rash, easily bruised or other evidence of haemorrhagic conditions. Three transfusion-transmission clusters have been reported; the first in Hong Kong, another in Singapore (where two clinical cases and one antibody seroconversion were documented) and lastly a case of transfusion-transmitted haemorrhagic fever in Puerto Rico. Infected individuals may donate blood since it has been estimated that 53–87% of dengue-infections are asymptomatic, and even individuals who develop symptoms will have a 1–2 day asymptomatic period. Puerto Rico, an island in the Caribbean and a US territory, experiences annual outbreaks. Research blood donation screening in Puerto Rico has documented the rate of dengue RNA in blood donors during the outbreak years of 2005, 2007, 2010, 2011 and 2012 at between 0·03% and 0·31%. The estimated number of viremic donations and the risk of dengue transfusion transmission have been modelled by the CDC. The resulting model output and the observed number of RNA-positive donors from blood donation screening show the same trend. Although routine screening by the American Red Cross occurs in Puerto Rico, the clinical efficacy of the intervention is unknown since the number of transfusion transmissions documented has been few. It is unclear why the number of transfusion transmissions is not higher in endemic areas considering the magnitude of dengue outbreaks and high viral loads associated with infected donors. Possible theories include an absence of effective hemovigilance in many of the countries impacted by dengue, inability to differentiate mosquito versus transfusion transmission, protection of secondary infection due to heterologous antibodies present in the transfused unit, immunosuppression in many recipients and possibly different clinical outcomes dependent on the route of infection (i.e. mosquito versus transfusion).
Sixty rodent samples were identified as PicoV positive, and 23 strains underwent genome sequencing (Additional file 1: Table S4). Rodent viruses from the genera Enterovirus, Hunnivirus, Mosavirus, Cardiovirus, Rosavirus, Kobuvirus, and Parechovirus were found in this study and showed 48.3–56.4%, 80.4–80.8%, 47%, 46.8–60.3%, 60.9%, 63–76.9%, and 43.7–87.3% RdRp aa identities with known members in each genus, respectively (Fig. 5b and Additional file 1: Table S11). Eight viruses formed lineages 1 and 2 close to the bat PicoV clade with 38.1–43.6%, 33.5–38.8%, and 48.2–56.7% aa identities with bat PicoVs in the P1, P2, and P3 regions, respectively. Two novel lineages 3 and 4 were identified with < 10.2–28.9% aa identities in the P1 region, 17.3–23.6% in the P2 region, and 21.8–28.4% in the P3 region compared with other PicoVs (Additional file 1: Table S10). Viruses closely related to known PicoVs of other hosts were found (e.g., rodent viruses related to human aichivirus, human rosavirus, and bovine hunnivirus).
More than 100 species of arbovirus that cause human/animal or zoonotic diseases have been identified.12 Four virus families, Togaviridae, Flaviviridae, Bunyaviridae and Reoviridae, contain most of the arboviruses that cause human/animal diseases.12 Arbovirus infections are not always clinically obvious and often resolve spontaneously after 1–2 weeks. However, some arboviral infections result in high fever, hemorrhage, meningitis, encephalitis, other serious clinical symptoms, and even death. Therefore, they cause a large social and economic burden. A summary listing the arboviruses associated with human diseases and their geographic distributions was published previously.21
Frugivorous, insectivorous or hematophagous bats worldwide have been studied for their role as reservoirs of infectious agents. Many viruses isolated from bats are able to cross the species barrier and infect humans, regularly causing severe diseases in humans (e.g., SARS, Ebola hemorrhagic fever, Nipah, rabies) (Table 2a). Most metagenomic studies targeting wildlife have been conducted on bats (Table 2b), as Calisher and collaborators reviewed in 2006, Wong and collaborators in 2007, Smith and Wang in 2013 or Luis et al. in 2013. Because “bat science” is a large and well-studied area in infectious diseases, this review will not focus more on this topic.
Many emerging infectious diseases are caused by zoonotic transmission, and the consequence is often unpredictable. Zoonoses have been well represented with the 2003 outbreak of severe acute respiratory syndrome (SARS) due to a novel coronavirus. Bats are associated with an increasing number of emerging and reemerging viruses, many of which pose major threats to public health, in part because they are mammals which roost together in large populations and can fly over vast geographical distances. Many distinct viruses have been isolated or detected (molecular) from bats including representatives from families Rhabdoviridae, Paramyxoviridae, Coronaviridae, Togaviridae, Flaviviridae, Bunyaviridae, Reoviridae, Arenaviridae, Herpesviridae, Picornaviridae, Filoviridae, Hepadnaviridae and Orthomyxoviridae.
The Reoviridae (respiratory enteric orphan viruses) comprise a large and diverse group of nonenveloped viruses containing a genome of segmented double-stranded RNA, and are taxonomically classified into 10 genera. Orthoreoviruses are divided into two subgroups, fusogenic and nonfusogenic, depending on their ability to cause syncytium formation in cell culture, and have been isolated from a broad range of mammalian, avian, and reptilian hosts. Members of the genus Orthoreovirus contain a genome with 10 segments of dsRNA; 3 large (L1-L3), 3 medium (M1-M3), and 4 small (S1 to S4).
The discovery of Melaka and Kampar viruses, two novel fusogenic reoviruses of bat origin, marked the emergence of orthoreoviruses capable of causing acute respiratory disease in humans. Subsequently, other related strains of bat-associated orthoreoviruses have also been reported, including Xi River virus from China. Wong et al. isolated and characterized 3 fusogenic orthoreoviruses from three travelers who had returned from Indonesia to Hong Kong during 2007–2010.
In the present study we isolated a novel reovirus from intestinal contents taken from one fruit bat ( Rousettus leschenaultia) in Yunnan province, China. In the absence of targeted sequencing protocols for a novel virus, we applied the VIDISCR (Virus-Discovery-cDNA RAPD) virus discovery strategy to confirm and identify a novel Melaka-like reovirus, the “Cangyuan virus”. To track virus evolution and to provide evidence of genetic reassortment PCR sequencing was conducted on each of the 10 genome segments, and phylogenetic analysis performed to determine genetic relatedness with other bat-borne fusogenic orthoreoviruses.
The Food and Agriculture Organization of the United Nations has recently estimated that the world equid population exceeds 110 million (FAOSTAT 2017). Working equids (horses, ponies, donkeys, and mules) remain essential to ensure the livelihood of poor communities around the world. In many developed countries, the equine industry has a significant economical weight, with around 7 million horses in Europe alone. The close relationship between humans and equids, and the fact that the athlete horse is the terrestrial mammal that travels the most worldwide after humans, are important elements to consider in the transmission of pathogens and diseases, amongst equids and to other species. The potential effect of climate change on vector ecology and vector-borne diseases is also of concern for both human and animal health.
With this Special Issue, which assembles a collection of communications, research articles, and reviews, we intend to explore our understanding of a panel of equine viruses, looking at their pathogenicity, their importance in terms of welfare and potential association with diseases, their economic importance and impact on performance, and how their identification can be helped by new technologies and methods. Beyond their potential risk to other species, including humans, equine viruses may also represent an interesting model for reproducing virus infection in the host species.
Dennis et al. contributed a review on African Horse Sickness (AHS). This disease, caused by the orbivirus AHS virus (AHSV), induces a very high mortality rate that can exceed 95% in its most severe form. This disease mostly occurs in southern African countries, but its transmission by Culicoides biting midges is of great concern in the current context of global warming and its consequence on displacement of vector populations. In the absence of treatment, prevention is essential, and Dennis et al. also provide a comprehensive review of the different vaccine strategies and technologies available and in current development against AHSV. While live attenuated and inactivated AHSV vaccines have played a role to reduce the impact and occurrence of AHS in affected areas, the number of AHSV serotypes in circulation and their lack of DIVA markers (differentiating infected from vaccinated animals) is a drawback that leads to the development of a new generation of vaccines, such as poxvirus-vectored or reverse genetics vaccines. Lecollinet et al. reviewed major viruses inducing encephalitis in equids and their growing importance as a threat to the European horse population. Amongst them, equid herpesviruses (EHVs) are some of the most frequently isolated equine viruses worldwide. The equine herpesvirus type 1 (EHV-1) is of particular interest to the equine industry because of the different forms of disease it can induce, from a mild respiratory infection to abortion, neonatal death, and myeloencephalopathy (EHM). A communication from Preziuso et al. and an article from Sutton et al. specifically focused on EHV-1 strain characterization in order to better understand EHV circulation in Italy and France. Different approaches were compared, from the single-nucleotide point (SNP) mutation in ORF30 (historically associated with abortive or neuro-pathogenic strains), to other ORF gene sequences and the newly described multilocus strain typing methods (MLST;). The MLST method is an interesting new approach for EHV-1 and a potential epidemiological tool that could provide an alternative until the development of more accessible EHV whole-genome sequencing methods. EHV-1 strain characterization by Sutton et al. allowed to conclude that the surge of EHV-1 outbreaks reported in France in 2018 was not linked to the introduction and/or circulation of a new EHV-1 strain in the French horse population. The origin of this crisis could be linked to a shortage of EHV vaccine and a subsequent reduced rate of EHV vaccination in the preceding years. Lecollinet et al. also reviewed less frequently isolated encephalitis viruses, which may be zoonotic, such as rabies virus, borna disease virus, and West Nile virus (WNV). In the case of WNV, both horses and humans are highly susceptible to viral infection through infected mosquitoes. Unprecedented circulations of WNV have been observed in several European countries in the last decade, with a potential role of climatic and environmental conditions. Both species are considered as dead-end hosts. However, the horse could be used as a sentinel species to monitor and control vector-borne virus activity. Other enzootic flaviviruses were also reviewed, such as Tick-Borne Encephalitis virus (TBEV) and Louping Ill virus (LIV). In Europe, vaccination is only available against some of these pathogens (i.e., EHV-1 and 4, Rabies virus, and WNV), which highlights the importance of surveillance. Taking into account that most of these viruses will induce similar clinical signs of disease, the development of discriminative diagnostic tools is also of increasing importance. Finally, the review presents some other vector-borne (mosquitoes or midges) equine encephalitis viruses, not currently circulating in Europe, from the Flaviridae family (i.e., the Japanese encephalitis virus (JEV), Saint Louis encephalitis virus (SLEV), and Murray Valley encephalitis virus (MVEV)) or the alphaviruses from the Togaviridae family (i.e., eastern, western, and Venezuelan equine encephalitis viruses; EEEV, WEEV, and VEEV, respectively). In relation to VEEV, Rusnak et al. presented systematic approaches for strain selection and propagation of virus and challenge material for the development and approval of a VEEV vaccine under the FDA Animal Rule and the different animal models available (rodents and non-human primates).
Altan et al. have used metagenomics to identify viruses in horses with neurological and respiratory diseases. The equine hepacivirus (EqHV) was detected in the plasma from several neurological cases. This virus, which was first reported in horses in 2012, was further investigated by Badenhorst et al., with a specific focus on its circulation in Austria and the potential role of mosquitoes in its transmission. The prevalence of EqHV in the Austrian horse population studies reached 45% (based on serological evidence), with around 4% of samples positive for EqHV RNA. No EqHV RNA was found in mosquitoes collected across Austria, raising questions about its methods of transmission. Some aspects of this particular question of EqHV transmission were treated by Pronost et al., who presented evidence to support a potential in utero transmission of EqHV from the mare to the foal, based on three positive clinical cases amongst 394 dead foals screened for the presence of EqHV RNA (prevalence of 0.76%). Altan et al. also detected two copiparvoviruses, the equine parvovirus-hepatitis (EqPV-H) and a new one named Eqcopivirus by the authors, with no specific and/or statistical association with disease. Equine parvovirus-hepatitis was also the subject of the article from Meister et al., which reported an EqPV-H infection occurrence in a quarter of the actively breeding Thoroughbred horse population from northern and western Germany. EqPV-H prevalence reached 7% and 35% (EqPV-H DNA positive detection and seroconversion, respectively). This study concerned mostly Thoroughbred brood mares, which represented 97% of the analyzed cohort. Concerning Thoroughbred stallions, Li et al. identified a new equine papillomavirus (EcPV9) in the semen from an Australian Thoroughbred stallion suffering from a genital wart. The clinical significance of this new equine papillomavirus remained to be determined and will require further investigation. A similar question was raised by Nemoto et al., who reported the first detection of equine coronavirus (ECoV) in Irish equids suffering from diarrhea. At five occasions, ECoV RNA was detected in feces from more than 400 equids with enteric diseases. However, the association with disease remains to be substantiated. While ECoV prevalence in Irish equids was 1.2% when measured by rRT-PCR in feces samples, evidence of ECoV infection was significantly higher when measured by serology in 984 serum samples from Dutch horses, 100 serums from Icelandic horses, and 27 paired serum samples from an ECoV outbreak in the USA. Zhao et al. developed and validated an S1-protein-based ELISA for this purpose. Seroprevalence ranged from 26% in young horses to nearly 83% in adults. The authors highlighted the potential use of this ELISA as a diagnostic test to confirm ECoV outbreaks, as a complement to feces samples analysis by qRT-PCR. The study from Back et al. shed some light on the potential role of equine rhinitis A virus (ERAV) infection in poor performance. This longitudinal study, which involved 30 Thoroughbred racehorses, significantly associated seroconversion to ERAV and subsequent failure to attend races. However, similarly to EqPV-H and ECoV infections previously reported in this Special Issue, a direct association of ERAV infection with clinical signs of disease could not be confirmed in this study.
Finally, Fatima et al. investigated the antiviral activity of the equine interferon-mediated host factors myxovirus (Mx) protein (eqMx1) against a range of influenza A viruses (IAVs). The authors highlight the potential protective role of eqMx1, which primarily targets the virus nucleoprotein (NP), against the transmission of new IAVs in horses (i.e., eqMx1 could only inhibit the polymerase activity of IAVs of avian and human origin but remained inactive against the equine IAVs tested). Introduction of a new IAV in the equine population is considered a rare event. In 1989, an equine influenza epizootic was reported in the Jilin and Heilongjiang provinces of northeastern China, with up to 20% mortality, which is quite high when compared with conventional equine influenza outbreaks. The IAV strain representative of this outbreak (i.e., A/equine/Jilin/1/1989) was closely related to an avian H3N8 IAV. The authors show that the IAV strain A/equine/Jilin/1/1989 bears two adaptive NP mutations that confer resistance to eqMx1. To date, equine influenza virus remains one of the most important respiratory pathogens of horses worldwide, with a potential damaging impact on the equine industry, as clearly illustrated in 2007 in Australia and in 2019 in Europe.
We hope this Special Issue helps to highlight the diversity of equine viruses and their importance, in terms of welfare and/or economic impact, to equids and humans.
Human microbiologic infections, known as zoonoses, are acquired directly from animals or via arthropods bites and are an increasing public health problem. More than two thirds of emerging human pathogens are of zoonotic origin, and of these, more than 70% originate from wildlife. In novel environments, viruses, particularly RNA viruses, can easily cross the species barrier by mutations, recombinations or reassortments of their genetic material, resulting in the capacity to infect novel hosts. Because of their adaptive abilities, RNA viruses represent more than 70% of the viruses that infect humans. When socio-economic and ecologic changes affect their environment, humans may encounter increased contact with emerging viruses that originate in wild or domestic animals.
Wolfe et al. in 2007 and Karesh et al. in 2012 described different stages in the switch from an animal-specific infectious agent into a human-specific pathogen. The key stage is the transition of a strictly animal-specific infectious agent (originating from wildlife or domestic animals) to exposed human populations, resulting in sporadic human infections (Figure 1). If the pathogen is able to adapt to its human host and acquire the means to accomplish an inter-human transmission, horizontal human-to-human transmission occurs and maintains the viral cycle. Sometimes, an intermediate host, such as a domestic animal, is the link between sylvatic viral circulation and human viral circulation. For example, some human infections originating from bats, such as Nipah, Hendra, SARS and Ebola viral infections, may involve intermediate amplification in hosts such as pigs, horses, civets and primates, respectively (Figure 1). Genetic, biologic, social, political or economic factors may explain a switch in viral host targets. For example, climate changes may influence the geographical repartition of vector arthropods, leading to new areas of the distribution of infectious diseases, like Aedes albopictus and Chikungunya infections in the Mediterranean. Morens et al. listed different key factors that may contribute to the emergence or re-emergence of infectious diseases, such as microbial adaptation to a new environment, biodiversity loss, ecosystem changes that lead to more frequent contact between wildlife and domestic animals or human populations, human demographics and behavior, economic development and land use, international travel and commerce, etc.. These patterns of transmission allow identifying different animals to follow in order to monitor the appearance of new or re-emerging infectious agents before its first detection in the human populations. Therefore, hematophagous arthropods, wildlife and domestic animals may serve as targets for zoonotic and arboviral disease surveillance, particularly because sampling procedures and long-term follow-up studies are more easily performed in these hosts than in humans.
Historically, classic viral detection techniques were based on the intracerebral inoculation of suckling mice or viral isolation in culture and the subsequent observation of cytopathic effects on cell lines. Later, immunologic methods, e.g., seroneutralization or hemaglutination, were used to detect viral antigens in various complex samples. These techniques were based on the isolation of viral agents. With the progresses of molecular biology, polymerase chain reaction (PCR)-based methods became the main techniques for virus discovery and allowed the detection of uncultivable viruses, but these techniques required prior knowledge of closely related viral genomes. Next-Generation Sequencing (NGS) techniques make it possible to sequence all viral genomes in a given sample without previous knowledge about their nature. These techniques, known as viral metagenomics, have allowed the discovery of completely new viral species. Because of their low cost, the use of NGS techniques is exponentially increasing.
The transmission of infections between humans occurs after a pathogen from a wild or domestic animal contacts with exposed human populations. The human exposures may or may not be mediated by the bite of bloodsucking arthropods. Surveillance programs may target wildlife, domestic animals or arthropods for emerging viruses before their adaptation to human hosts.
Each year in the United States, there are approximately 76 million cases of food-borne illness, including 325,000 hospitalizations and 5,000 deaths. In an estimated 2 to 3% of these cases, chronic sequelae develop. These sequelae include renal disease, cardiovascular diseases, gastrointestinal disorders, neural disorders, and autoimmune disease. The estimated cost of food-borne illness in the United States is $23 billion annually. Mishandling of food is believed to be responsible for 85% of all outbreaks of food-borne disease in developed nations, primarily due to a lack of education. Food-borne pathogens [see Additional File 3] are also important because they represent one of the largest sources of emerging antibiotic-resistant pathogens. This is due in part to the administration of sub-therapeutic doses of antibiotics to food-producing animals to enhance growth. For example, certain strains of Salmonella show resistance to eight or more antibiotics. Studies have shown that antibiotic resistance in Salmonella cannot be traced to antibiotic use in humans, suggesting that antibiotic use in animals is the primary cause of resistance.
While much is known about the major microbes responsible for diseases, there are still many undiagnosed cases of infectious disease. It has been estimated that as many as three-fifths of the deaths from acute gastroenteritis per year in the United States are caused by an infectious organism of unknown etiology. Four of the major causes of food-borne infections (Campylobacter jejuni, Escherichia coli O157:H7, Listeria monocytogenes, and Cyclospora cayetanensis, Figure 2) were only recently recognized as causes of food-borne illness.
Arboviruses are transmitted between arthropods (mosquitoes, ticks, sandflies, midges, bugs…) and vertebrates during the life cycle of the virus.8 Many arboviruses are zoonotic, i.e., transmissible from animals to humans.9,10 As far as we are aware, there are no confirmed examples of anthroponosis, i.e., transmission of arboviruses from humans to animals.9,10 The term arbovirus is not a taxonomic indicator; it describes their requirement for a vector in their transmission cycle.11,12 Humans and animals infected by arboviruses, may suffer diseases ranging from sub-clinical or mild through febrile to encephalitic or hemorrhagic with a significant proportion of fatalities. In contrast, arthropods infected by arboviruses do not show detectable signs of sickness, even though the virus may remain in the arthropod for life. As of 1992, 535 species belonging to 14 virus families were registered in the International Catalogue of Arboviruses.12 However, this estimate is continuously increasing as advances in virus isolation procedures and sequencing methods impact on virus studies. Whilst many current arboviruses do not appear to be human or animal pathogens, this large number of widely different and highly adaptable arboviruses provides an immense resource for the emergence of new pathogens in the future.
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.
The following primer and probe sets are available from Genetic Signatures: pan-flavivirus, pan-alphavirus and pan-dengue (FA001), Zika virus (FA002-A), West Nile virus (FA002-B), Yellow Fever Virus (FA002-C), Tick-borne encephalitis virus (FA002-D), St Louis encephalitis virus (FA002-E), Murray Valley encephalitis virus (FA002-F), dengue-1 (FA003-A), dengue-2 (FA003-B), dengue-3 (FA003-C), dengue-4 (FA003-D), chikungunya (FA004-A), Ross River virus (FA004-B), Barmah Forest virus (FA004-C), Eastern equine encephalitis virus (FA004-C), Western equine encephalitis virus (FA004-D) and Venezuelan equine encephalitis virus (FA004-E).
Serum or plasma samples from patient with suspected DENV infection were collected at Port Vila Central Hospital, Efate, Vanuatu between December 2016 to March 2017. 100 μL of material along with 5 μL of EPC was processed using the EasyScreen Sample Processing Kit, SP001 (Genetic Signatures, Sydney, Australia) according to the manufacturer’s instructions. After bisulphite conversion the samples were extracted on the GS-mini automated nucleic extraction platform using the EasyScreen Sample Processing Kit, SP006 (Genetic Signatures, Sydney, Australia) according to the manufacturer’s instructions.
IgG antibodies to the novel bunyavirus were detected in 80 of 285 acute-phase serum samples from patients with FTLS (Table 5). Of 95 patients from whom paired acute- and convalescent-phase sera were available, 52 had seroconversions and 21 had greater than 4-fold increases in antibody titer to the virus. Six had less than a 4-fold increase in antibody titer to the virus, but all paired sera tested positive. Sixteen patients tested negative to the virus, suggesting that some non-FTLS patients with similar symptoms were included in this study, a situation that is not surprising given that FTLS is a newly emerging disease. The acute-phase sera of four patients from whom the virus was isolated tested negative for IgG antibody to the virus. All convalescent sera obtained 2 months later from the same four patients contained IgG antibody to the virus. None of the 130 sera from patients with respiratory diseases or healthy subjects had detectable antibody.
Since 2007, there has been an increase in reported cases of FTLS in Xinyang City, Henan Province. These patients were tentatively diagnosed as having A. phagocytophilum infection. However, only a few (8.4%, 24/285) such patients had evidence for A. phagocytophilum infection, and none of the 285 patients tested positive for the many other pathogens capable of causing similar clinical and laboratory manifestations that were also investigated. These findings suggested novel infectious agents, including viruses.
Traditionally, virus culture is very important for identifying an unknown viral infection. Before performing the Illumina sequencing strategy, we attempted viral and rickettsial culture with DH82 and BHK cell lines, but the lack of an obvious CPE led us to initially abandon this approach. Here, mass sequence data obtained by Illumina sequencing revealed four virus families that appeared only in FTLS patient sera. Among these four virus families, viruses from the Parvoviridae and Bunyaviridae families reportedly can cause signs of FTLS and be transmitted by arthropods. However, only one sample from a pool of ten samples tested positive for bocavirus by PCR, suggesting that bocavirus from the Parvoviridae is not likely involved in FTLS. For viruses in the Bunyaviridae family, the incidence of infection is closely linked to vector activity. For example, tick-borne viruses are more common in the late spring and late summer when tick activity peaks. Human infections with certain Bunyaviridae, such as Crimean-Congo hemorrhagic fever virus, are associated with high levels of morbidity and mortality. Considering the tick-bite history of many FTLS patients, we focused on Bunyaviridae family viruses.
The entire Bunyaviridae family contains more than 300 members arranged in four genera of arthropod-borne viruses (Orthobunyavirus, Nairovirus, Phlebovirus and Tospovirus) and one genus (Hantavirus) of rodent-borne viruses,. The Phlebovirus genus currently comprises 68 antigenically distinct serotypes, only a few of which have been studied. The 68 known serotypes are divided into two groups: the Phlebotomus fever group (the sandfly group, transmitted by Phlebotominae sandflies) comprises 55 members, and the Uukuniemi group (transmitted by ticks) comprises the remaining 13 members. Of these 68 serotypes, eight are linked to disease in humans, including the Alenquer, Candiru, Chagres, Naples, Punta Toro, Rift Valley fever, Sicilian, and Toscana viruses. Phleboviruses have tripartite genomes consisting of a large (L), medium (M), and small (S) RNA segment.
In screening for unknown viruses, species hits alone likely carry little weight. Thus, we used all sequences in the family Bunyaviridae for our analysis. A 168-bp fragment of the polymerase gene with the lowest E-value and high sequence identity was used as the sequence of the unknown virus. This virus sequence was detected in all 10 pooled samples, indicating that the virus is involved in FTLS. After detecting a possible novel bunyavirus through high-throughput Illumina sequencing, we inoculated Vero cell lines, which are known to be sensitive to phleboviruses, with sera from six positive patients and were subsequently able to detect the virus by RT-PCR,. Although the CPE was modest, RT-PCR confirmed the infection. Genome sequencing was performed and a phylogenetic analysis of the genome sequence showed that this virus clustered into the Phlebovirus branch, but was divergent from other known phleboviruses. These results confirm the novelty of this virus within the Phlebovirus genus of the family Bunyaviridae
. Furthermore, virus size and propagation in cells were similar to that of the bunyaviruses.
PCR and serological tests were performed to further test the causal link between the new virus and FTLS. Although we have not completely fulfilled Koch's postulates, evidence implicating this new bunyavirus in the outbreak of the disease among patients with FTLS is compelling.
In view of the fact that the disease is caused by a novel bunyavirus, and taking into account that the disease was first discovered in Henan (HN), we propose the name "Henan Fever" for the FTLS disease cause by the novel virus (proposed name “Henan Fever Virus” [HNF virus]). Since the submission of this manuscript, a bunyavirus was identified as the cause of FTLS in Chinese patients from other regions of China, and the authors have named this virus “SFTSV” to indicate that it is the cause of severe fever with thrombocytopenia syndrome. After release of the GenBank sequences referred to in the Yu paper, we compared the sequences of SFTSV with those of FTLSV and found that they were nearly identical (>99% identity). As we first identified the syndrome in 2007 and described the presence of the virus in patients between 2007 and 2010, we suggest that the name “HNF virus” should take precedence. The most distinctive feature of the current work includes the use of an unbiased metagenomic approach for viral pathogen discovery that facilitated the rapid creation and implementation of standard culture, serological, and molecular diagnostic approaches. However, there are other differences between the results described here and those reported by Yu et al; notably, we observed slight, but distinctive, CPE in Vero cells. The reason for the failure to observe CPE in Vero cells infected with the “SFTSV” bunyavirus, whose genome is nearly identical to that of bunyavirus isolated from our FTLS patients, is unclear. Perhaps this reflects the fact that the ensuing CPE is not dramatic. Alternately, this could indicate the existence of distinct viral strains that vary in pathogenicity, virulence, and possibly even disease manifestations. This is an area of active study in our laboratories.
The discovery of this new virus will assist in the rapid diagnosis of this disease and help to distinguish it from other diseases caused by pathogens such as A. phagocytophilum, E. chaffeensis, Crimean-Congo hemorrhagic fever virus, Hantavirus, dengue virus, Japanese encephalitis virus, and Chikungunya virus. Furthermore, the availability of the new virus will facilitate the future development of new therapeutic interventions, such as vaccines and drugs.
Venezuelan equine encephalitis virus (VEEV) is one of the most neglected viruses among biowarfare agents. It is classified as a Category B biothreat pathogen by National Institute of Allergy and Infectious Diseases (NIAID), USA, due to its high dissemination rate, minimal infectious dose to induce disease in human, and the requirement for specific and enhanced diagnostic capacities. VEEV causes disease with symptoms ranging from influenza-like illness to more severe illnesses including myalgia, arthralgia, and neurological disorders that can lead to lethal encephalitis in susceptible hosts. All these characteristics made VEEV an attractive candidate for weaponization (1). VEEV was first isolated from an infected horse brain during a VEEV outbreak in Venezuela in 1938 followed by major outbreaks in Venezuela and Columbia in 1960s (2), infecting thousands of people and animals. Despite its high contagiousness, VEEV has drawn little public attention due to only sporadic outbreaks occurring in Central and South America since 1995. Other reasons for being neglected may be a low mortality rate in humans (<1%) while as high as 90% in horses, as well as no reports of VEEV outbreaks in the US since the only epidemic outbreak in Texas in 1971 (3). However, the spillover of VEEV infection from infected horses to humans during epidemics remains a concern.
VEEV is an arthropod-borne epizootic RNA virus, belonging to the Togaviridae family, genus Alphavirus, and is maintained within lower animals (rodents) and mosquitos (4). The transmission of VEEV is primarily mediated by mosquitoes, where it replicates in salivary glands and is passed to hosts, such as human and horses with overt symptoms (5, 6). Moreover, aerosolized VEEV can be directly disseminated and can infect humans or susceptible experimental animal models, causing encephalitis, and possibly limb paralysis (7–9). Historically, since the late 1930s the former Soviet Union regarded VEEV as an operational biological weapon to incapacitate the rear services and reinforcement behind the front line, leading to the spread of the disease among infected individuals with flu-like symptoms that are difficult to distinguish from epidemic influenza outbreaks. Weaponized VEEV was not expected to kill the soldiers, but cause panic and ultimately maim the military targets (10). Notably, there are no effective antiviral agents available and approved by the US Food and Drug Administration (FDA) and only supportive treatment is available for humans. As a result, it is important to establish a preventive surveillance system based on prompt diagnosis methods.
Viral stability is an essential factor for diagnosis of VEEV to confirm the presence of virions or viral RNA in a clinical sample. The current diagnostic approaches to confirm VEEV infection in humans or horses rely on direct detection of viral nucleic acids in serum or spinal fluid samples during the acute-phase of infection using reverse-transcription PCR (RT-PCR) (11) and ELISA for VEEV-specific IgM (12). However, despite high sensitivity of the above-mentioned methods, false negative results can be obtained if samples have been collected during the initial asymptomatic phase of infection where the viral load is low (13–15). Moreover, the necessity of extra steps of plasma or serum preparation complicates fast virus identification and diagnostic. Therefore, virus stabilization directly in collected sample without extra preparation steps is highly desirable. Ideally, supplementation of the blood collection device with some type of stabilization agent that can minimize pathogen loss during sample transportation and storage at ambient temperatures is appealing. In view of these challenges, Nanotrap® (NT) particles were evaluated for their ability to stabilize VEEV. NT particles are hydrogel polymer particles comprised of N-isopropylacrylamide (NIPAm), allylamine (AA), and crosslinked with N,N′-methylenbisacrylamide (BIS). These particles are functionalized with various dye affinity baits that facilitate capture and retention of analytes from complex biological matrixes (such as blood, saliva, nasal swabs and urine) and concentrate them into a smaller volume (16–20). Previous work with NT particles has shown the benefit of their use in the enrichment of infectious virus and viral genomic material of Rift Valley fever virus (RVFV), coronavirus, influenza virus, and respiratory syncytia virus (19, 21).
In this study, we sought to apply new magnetic NT particles that consist of NIPAm copolymers functionalized with reactive red 120 to evaluate the efficacy of preservation of infectious VEEV, viral RNA, and VEEV capsid protein in whole blood samples at ambient and elevated temperature as well as at low and high humidity conditions. Our results indicate that: (i) magnetic NT particles enhance preservation of infectious VEEV in whole human blood at 40°C; (ii) NT maintain significantly higher levels of VEEV RNA in whole human blood at 40°C; (iii) NT retain their functional activity at both normal and elevated humidity conditions and significantly preserve VEEV infectivity in such an environment; and (iv) blood samples from VEEV TC-83 infected animals are better protected from capsid protein degradation if they are incubated with NT. Our results demonstrate for the first time the capability of NT particles to stabilize virus in blood at elevated temperatures, the direct interaction of NT particles with VEEV (via transmission electron microscopy), and the utility of NT particles with viral clinical samples.
Infections with multiple other viruses such as measles and mumps (Paramyxoviridae family), and rubella (Togaviridae family), have been linked to autoimmune disorders. Specifically, mumps and rubella infections were linked to the onset of type 1 diabetes. Both viruses have the ability to infect and grow in beta cells. Further, both viruses result in CNS demyelination disease. Viruses from the Flaviviridae family, such as Zika virus (ZIKV) and dengue virus (DENV) have been also associated to autoimmune disorders, including the recently reported ZIKV-induced Guillain–Barré syndrome and DENV-induced SLE and lupus nephritis. Human T-lymphotropic virus type 1 (HTLV-1) from the Retroviridae family has been associated with CNS autoimmunity, causing myelopathy/tropical spastic paraparesis. Several recent studies aimed to explain the contribution of viral infections in CNS disorders. It was suggested that antiviral immune response cross-react with human NMDA receptors (2A subunit), which are responsible for excitatory glutamatergic transmission and are ion channel proteins found in nerve cells. Pentapeptide similarity between the NMDA 2A receptor and viral proteins have been previously reported. Using the same mechanism, other peptide commonalities with high degree of pentapeptide sharing were found between viral peptides and the human distal-less homeobox (DLX) transcription factors expressed during early fetal neurodevelopment (DLX1, DLX2, DLX5, and DLX6). Analysis of the brain-specific DLX self-antigens pentapeptide revealed matching with several viruses that have been related to neurological disorders including rubella and herpesviruses. Collectively, these studies support the assumption that viral infections may relate to CNS disorders through autoimmune cross-reactions caused by molecular mimicry.
In a recent work by Kanduc, peptide sharing analysis of five common viruses (Borna disease virus, IAV, measles, mumps, and rubella) in comparison to human proteome revealed an unexpected massive viral human peptide cross-reactivity. The author explained this finding in the light of the viral eukaryogenesis hypothesis, which describes that the first eukaryotic cell had evolved from an archaeal ancestor of the eukaryotic cytoplasm, a bacterial ancestor of the mitochondria, and a viral ancestor of the nucleus. Importantly, the clinical implication of this high viral/human peptide sequence similarity confirms the significant contribution of molecular mimicry as a mechanism in viral induced autoimmunity.
Considering the available data from epidemiological and experimental animal studies, there is a wide range of viruses that are suspected to initiate an autoimmune response, despite the lack of a clear mechanistic explanation to this phenomenon in most of the cases. Table 1 summarizes studies reporting viral induced autoimmunity in different organisms along with proposed mechanisms. Importantly, it is clear that there is no single factor responsible for triggering autoimmunity. It seems that the development of autoimmune diseases following viral infections is a multifactorial process that can be affected by different variables. Moreover, determining whether viral infection can lead to autoimmunity or protect from certain immune disorders such as diabetes and CD, depends on multiple factors, including virus strain, genetic predisposition, host immune response, infectious dose, and time of infection.
In the developing world, nearly 90% of infectious disease deaths are due to six diseases or disease processes: acute respiratory infections, diarrhea, tuberculosis, HIV, measles, and malaria [see Additional File 1]. In both developing and developed nations, the leading cause of death by a wide margin is acute respiratory disease. In the developing world, acute respiratory infections are attributed primarily to six bacteria: Bordetella pertussis, Streptococcus pneumonia, Haemophilus influenzae, Staphylococcus aureus, Mycoplasma pneumonia, Chlamydophila pneumonia, and Chlamydia trachomatis. These bacteria belong to four different taxonomic classes and illustrate how similar parasitic lifestyles can evolve in parallel within unrelated bacterial species (Figure 2). Major viral causes of respiratory infections include respiratory syncytial virus (Figure 5), human parainfluenza viruses 1 and 3 (Figure 5), influenza viruses A and B (Figure 5), as well as some adenoviruses (Figure 4).
The major causes of diarrhoeal disease in the developing and developed world have significant differences due to the great disparity of availability of pure food and water and the general nutritional and health status of the populations. Important causes of diarrhoeal disease in the developing world are those that tend to be epidemic, especially Vibrio cholera, Shigella dysenteriae, and Salmonella typhi. These organisms are gammaproteobacteria (Figure 2) that use many different metabolic pathways to ensure their survival in a wide range of environments. In the United States there is a much lower incidence of diarrhoeal disease overall, and a relatively greater impact of direct human-to-human infectious transmission. The most important causes of diarrhoeal disease in the United States are bacteria such as Escherichia coli, Campylobacter species, Clostridium difficile, Listeria monocytogenes, Salmonella enteritidis, and Shigella species (Figure 2); viruses, such as Norwalk virus (Figure 6) and rotaviruses (Figure 7); and parasites such as Cryptosporidium parvum, Cyclospora cayetanensis, Entamoeba histolytica, Giardia lamblia, while microsporidia are responsible for a smaller number of cases (Figure 3).
Infectious disease agents important to the public health in the U.S. are monitored by the CDC and listed in Additional File 2 [see Additional File 2]. There are no set criteria for inclusion on the notifiable disease list; rather, the list is created by the CDC in cooperation with state health departments. As diseases occur less frequently and new diseases emerge, the notifiable disease list changes. The list provides links to case definitions of each disease, including the etiological agent(s) responsible. In cases where the etiological agent was not listed or was unspecific (i.e. Brucella spp.), further research was done to determine an etiological agent and this information is in Additional File 2 [see Additional File 2].
Viral infections are a major trigger of autoimmunity. Virus-induced autoimmunity is a multi-directional process. Current data suggests that viruses can initiate autoimmunity via several pathways including molecular mimicry, epitope spreading, bystander activation and/or immortalization of infected B cells. To the contrary, a growing evidence is supporting the protective role of viruses against autoimmunity, where viral infections lead to the activation of regulatory immune responses, consequently suppressing the development of autoimmune reactions. This dual effect of viral infections on autoimmunity is orchestrated by different host, viral and environmental factors. Accordingly, further epidemiological and molecular research is needed to gain insights about the interplay between viral infections and host autoimmune responses, and to provide a clear mechanistic description on how a viral infection can trigger autoimmunopathies.