The application of NGS to the unbiased mass sequencing and bioinformatic analysis of total nucleic acids extracted from biological samples obtained from a wide range of sources has led to an explosive increase in the number of complete or near-complete viral genomes. For example, a recent study using NGS to sequence the transcriptomes of arthropods representing 70 species from four classes (Insecta, Arachnida, Chilopoda, and Malacostraca) identified 112 novel viruses, many of which are represented by complete or near-complete genomes. The novel viruses encompass the entire taxonomic diversity of previously known families and/or genera of (-) ssRNA viruses and include divergent viruses with entirely novel and unusual genome architecture. Similarly, sequence analysis of the transcriptomes of animals of more than 220 species sampled across nine metazoan phyla (Arthropoda, Annelida, Sipuncula, Mollusca, Nematoda, Platyhelminthes, Cnidaria, and Echinodermata), as well as chordates of the subphylum Tunicata (salps and sea squirts), resulted in the discovery of 1445 RNA viruses, mostly represented by complete or near-complete genomes. Based on phylogenetic analysis of RNA polymerase (RdRp) domain sequences, the novel viruses included clades representing many established families of plant and animal RNA viruses, as well as at least five clades that are so divergent that they are considered as likely new virus families or orders. Also, the sequencing of transcriptomes of gut, liver, and lung or gill tissue of fish, reptiles, amphibians, and birds identified 214 novel vertebrate-associated viruses, representing every family or genus of RNA virus associated with vertebrate infection, including those containing important human pathogens (orthomyxoviruses, arenaviruses, and filoviruses). These and other similar studies have heralded a new era in virology, revealing new dimensions in viral biodiversity and providing largely unexpected insights into the deep evolutionary history of viruses. However, only a minor subset of newly discovered viruses has been subject to full phenotypic characterization which can provide critical and fundamental insights into their biology and virus-host interactions, ultimately transforming our understanding of the evolutionary forces that shape the virosphere and disease emergence. Realistically, as important as these discoveries are to the advancement of science, very few of the viruses will ever cause human disease or influence the global economy.

This mass sequencing approach, which has been called viral metagenomics, can also be applied in a more targeted way to identify viruses in clinical cases of diseases of unknown etiology or to survey for potentially novel zoonotic viruses that may represent a significant risk of transmission to humans. Indeed, NGS is being used increasingly in conjunction with real-time PCR as a front-line tool in medical and veterinary settings for rapid detection and identification of exotic or unknown emerging viruses. For example, in 2009, an outbreak of acute hemorrhagic fever occurred in Mangala, Democratic Republic of Congo (DRC), involving three human cases, two of which were fatal. As no positive diagnosis was obtained using real-time PCR for known viral hemorrhagic fevers in Africa, NGS was conducted on acute phase serum collected from the surviving patient, revealing the near-complete sequence of a novel rhabdovirus, Bas-Congo virus (BASV; species Bas Congo tibrovirus). Although the disease was attributed by the investigators to the novel virus, no isolate was obtained and there was no evidence of neutralising antibodies in 43 serum samples from undiagnosed hemorrhagic fever cases or 50 random serum donors from the DRC. Subsequently, NGS of blood collected from healthy individuals from Nigeria identified two related viruses, Ekpoma virus 1 (EKV-1; species Ekpoma 1 tibrovirus) and Ekpoma virus 2 (EKV-2; species Ekpoma 2 tibrovirus), and a serological survey indicated that antibodies to these or similar viruses occur commonly in healthy humans in Nigeria. However, once again, neither virus was isolated. Interestingly, several other tibroviruses had previously been isolated from healthy cattle or biting midges (Culicoides spp.) in Australia and Florida. None have been associated with either disease in livestock or the infection of humans. These and other studies raise important issues regarding the significance of NGS data, even when providing complete or near-complete viral genome sequences, when investigating disease etiology. Most viruses can cause asymptomatic infections and many newly discovered viruses may be benign in their natural host. Therefore, in the absence of a virus isolate which can be used for experimental studies, establishing a causal association with disease based only on detection of the viral genome should be approached cautiously.

Nipah is paramyxovirus of the genus Henipavirus (family Paramyxoviridae) with a high fatality rate (69). Infection in humans usually causes severe encephalitic and respiratory disease (70). After inoculation with Nipah virus (NiV), Syrian hamsters also develop characterisitic neurological disease (12). Similar to symptoms after human infection, pathological lesions are the most severe and extensive in the hamster brain and viral antigen and RNA can be detected in neurons (11), lung (71), kidney, and spleen (11). The Syrian hamsters in the majority of NiV infection studies are treated by intraperitoneal (IP) injection or intranasal (IN.) delivery and these models have revealed that different inoculation method can cause diverse pathological responses (11). In Wong's work, IP injection of NiV in Syrian hamsters caused primarily neurological disease, while IN delivery developed neurological symptoms as well as labored breathing due to lung infection in the final stages of disease (11). Disease progression is usually much rapid and the time to death post-infection is shorter following intraperitoneal rather than intranasal inoculation (72). Since the Syrian hamster has shown suitability for studying NiV infection, it was further used to study the viral transmission (73–75), demonstrating that Nipah virus is transmitted efficiently via direct contact and inefficiently via fomites, but not via aerosols. Regarding the use of these models for development of disease treatment and prophylaxis, recent studies have shown that pretreatment with Poly(I)-poly(C12U) can significantly decrease the mortality caused by NiV infection of Syrian hamster (76). In addition, the model was used as a platform for evaluation of vaccines for NiV (77–80). Walpita et al. discovered purified NiV-like particles (VLP) can protect the Syrian hamster using either multiple-dose or single-dose vaccination regimens followed by NiV challenge (81).

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.

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.

Coronaviruses (CoVs) prior to the SARS outbreak were only known to be the second cause of the common cold after rhinoviruses. At least four different species can cause mild, self-limiting upper respiratory tract infections in humans: alphacoronaviruses HCoV-229E and HCoV-NL63, and betacoronaviruses HCoV-HKU1 and HCoV-OC43. More recently, two more additional pathogenic human-CoV were identified: Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV).23 SARS-CoV was first identified in China in February 2003, and 4 months later, >8000 cases had been reported with about 800 deaths in 27 different countries worldwide.24 SARS-CoV has a wide host range and it is associated with wildlife meat industry. The natural history of the virus involves bats as primary hosts that then transmitted it to the intermediate amplifying hosts – as mask palm civets and raccoon dogs – that then could spread it to humans.23,25 Human-to-human transmission follows and can lead to large numbers of infected patients and is considered the main route of transmission in large-scale epidemics.9

MERS-CoV is phylogenetically related to SARS-CoV and share with SARS-CoV the origin in bats.23,26,27 Several CoVs have been identified in insectivorous and frugivorous bat species in various countries, indicating that bats may represent an important reservoir of these viruses.23 MERS-CoV was first identified in Saudi Arabia in 2012 and then spread to other countries causing hundreds of deaths.26,28 Clinical features of MERS-CoV are similar to SARS-CoV, although this virus has also been associated with several extrapulmonary manifestations, such as severe renal complications. Recent studies have indicated that dromedary camels may be the intermediate hosts and potential source of the virus for humans.26,29 In addition, the first experimental infection of bats with MERS-CoV has been described. The virus maintains the ability to replicate in the host without clinical signs of disease, supporting the general hypothesis that bats are the ancestral reservoir for MERS-CoV.30 Human-to-human transmission has also been reported. Based on epidemiological data, both animal-to-human and human-to-human transmission are considered to be important elements in MERS outbreak.26

The Flaviviridae is a large family of positive-strand RNA viruses, that comprises four genera: Flavivirus, Pegivirus, Pestivirus, and Hepacivirus. The Flavivirus genus consists of more than 70 viruses, many of which are arthropod-borne human pathogens that cause a variety of clinical diseases, ranging from asymptomatic to mild fever to more severe diseases including encephalitis and hemorrhagic fever [1, 2] Most flaviviruses are transmitted through the bite of an infected arthropod vector, mainly Aedes genus (Aedes aegypti and to a lesser extent, Aedes albopictus) and Cluex mosquitos, and most were once maintained by animal reservoirs in sylvatic transmission cycles. Many flaviviruses, however, such as dengue virus, yellow fever and Zika virus, are now principally maintained by mosquito-borne transmission with a possible human-to-human transmission through transfusion of infected blood or transplantation of infected tissue.

Some flaviviruses can cause globally significant vector-borne diseases with a substantial public health impact such as dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), Zika virus (ZIKV) and yellow fever virus (YFV). Other members of the Flaviviridae family that have a more regional impact include Murray Valley encephalitis virus (MVEV) in Oceania, St. Louis encephalitis virus (SLEV) in North America, and tick-borne encephalitis virus (TBEV) in Europe. Over the past few decades, many of these flaviviruses have re-emerged for a range of reasons including decreases in mosquito control efforts, rapid changes in climate and vector's demography, dense urbanization, population growth and globalization with increased transportation and trade activities. Examples include the geographic spread of DENV throughout the tropical world; JEV throughout south Asia, Australasia and the Pacific; ZIKV into South and Central America; YFV into the Americas and the invasion of WNV into much of North America [1, 5].

It is estimated that there are over 390 million DENV infections per year, of which 96 million manifests clinically with varying degrees of severity and 3.9 billion people in 128 countries are at risk of infection. Similarly, high incidence rates for symptomatic cases of JEV were reported over the past three decades to reach 2.4 per year per 100,000 population. Epidemic waves of YFV are projected to result in 30,000 to 200,000 clinical cases per year with case-fatality rates ranging from 2 to 15% [9–12]. WNV, first appeared in the northeastern USA in 1999, are spread presently across much of the USA and southern Canada. For example, in 2015, the CDC reported 2,175 cases of WNV, of which 1,616 (74%) were hospitalized and 146 (7%) died. In the developing world, WNV incidence is likely to be underestimated due to political, psychological, and economic barriers to reporting [12, 14].

Although most human flavivirus infections are asymptomatic or have an undifferentiated febrile illness, a small percentage of affected individuals develop acute fever that can progress to severe clinical manifestations such as hemorrhage, vascular leakage and encephalitis. Currently, our knowledge of the host-related factors that influence the pathogenesis of severe disease is inadequate to allow prediction of who will develop severe clinical illness. However, some mechanisms and etiological factors underlying inter-individual variations in response to flavivirus infections have been identified. Interactions of virus-encoded proteins with human innate immune pathways; the effect of host-cell surface molecules in virus binding and entry; the role of viral protein nuclear localization in the host cell response; and the flavivirus replication dynamics within multiple immune systems have all been considered as host-pathogen interaction events that may regulate viral virulence or attenuation and the subsequent disease severity. Over the past few years, however, host-related factors such as preexisting chronic conditions, e.g., cardiovascular diseases, diabetes, obesity, and asthma have received attention as predictors for increased risk of progression to severe flavivirus infection [19–21]. Recent studies have raised the proposition that cardiovascular disease, stroke, diabetes, respiratory diseases and renal disorders may contribute, together with old age, to severe clinical manifestations of dengue [19, 20]. A few studies of WNV and JEV infections, and responses to YFV vaccination, have also explored the role of chronic comorbidities in the prognosis of infections. Given the lack of specific medical treatment for flavivirus diseases, effective public health surveillance for vector-borne infections together with continuing vector control efforts will be critical to preventing infection. However, elucidating the impact of comorbidities to the severity of disease when infection occurs will be critical to identifying vulnerable populations, to whom effective interventions protocols and individually-tailored clinical monitoring practices should be particularly targeted.

The objective of this study is to systematically review the existing literature on the prevalence of the most common non-communicable comorbidities related to the cluster of metabolic syndromes-associated diseases, such as diabetes mellitus, heart diseases, hypertension, asthma, stroke and obesity in flavivirus infections and to evaluate the difference of their prevalence in severe vs. non-severe clinical outcomes to infection. Identifying and characterizing associations between comorbidities and severity of flavivirus infections will be significant factor in designing public health measures that aim to prevent the severe outcomes of infection.

YFV is an arthropod-borne virus of the genus Flavivirus (family Flaviviridae) and has high morbidity and mortality rates in regions of sub-Saharan Africa and South America (53). It was one of the first viruses of humans to be identified, isolated, propagated in vitro and studied by genomic sequencing (54). The study of infection mechanism of YFV has historically been hindered by the lack of appropriate small animal model and non-human primate (NHP) models have typically been used. More recently, several research groups have generated animal models using Syrian hamsters that can be successfully infected with YFV (55–58). McArthur et al. reported adapted viral strains (Asibi/hamster p7) allow the reproduction of yellow fever disease in hamsters with features similar to the human disease (59). Further, studies have also shown that infection of Syrian hamster results in immune responses that correspond to those observed in infected humans, with marked increases in IFN-γ, IL-2, TNF-α in the spleen, kidney, and heart, but reduced levels of these seen in the liver. In addition, these studies found increased levels of IL-10 and reduced levels of TGF-β in the liver, spleen, and heart in early and mid-stages of infection (60). Syrian hamster can be used both to study the pathogenesis of the YFV infection, and to validate antiviral drugs and antiviral therapies. Recent findings have shown that treatment with the anti-viral compounds 2′-C-methyl cytidine (61), T-1106 (62), IFN alfacon-1 (63), and BCX4430 (64) pre- and post-YFV exposure can significantly improve Syrian hamster survival. In a study by Julander et al. immunization with DEF201, an AdV type-5 vector expressing IFN alpha (IFN-α), can effectively reduce the viral titer in hamster's liver and serum post-YFV infection (65). Immunoprophylaxis with XRX-001, a vaccine containing inactivated yellow fever antigen with an alum adjuvant, can elicit high titers of neutralizing antibodies in vivo to protect Syrian hamsters from YFV infection (66, 67). Interestingly, Xiao et al. (67) and Tesh et al. (68) demonstrate that prior exposure of Syrian hamsters to heterologous flaviviruses reduces the risk of YFV infection.

Paramyxoviridae constitute a wide viral family that includes human and animal pathogens. Several bat-borne paramyxoviruses have been recognized such as parainfluenza type 2 virus, Mapuera, Menangle and Tioman viruses and two infectious agents of emerging diseases, such as Nipah and Hendra viruses.20

Nipah and Hendra viruses, classified as the genus Henipavirus, are capable of causing severe, potentially fatal diseases in humans.20 Fruit bats of the Pteropus genus are the common reservoir hosts of the Nipah and Hendra viruses.20

Nipah virus (NiV) first emerged in 1998 in Malaysia, causing an outbreak of respiratory illness and encephalitis in pigs.21 Pig-to-human transmission of Nipah virus – associated with severe febrile encephalitis – was described and it was thought to occur through close contact with infected animals. Although uncommon, human-to-human transmission of virus was also described.21 In two other outbreaks in Bangladesh and India, an intermediate animal host was not identified, suggesting bat-to-human and human-to-human transmissions.

Hendra virus (HeV) causes a fatal respiratory disease in both humans and horses.20,22 Several outbreaks of HeV have occurred in Australia. Horse is the intermediate host and the virus is likely transmitted via ingestion of feed, pasture or water contaminated with urine, saliva and feces of infected bats. Horse-to-human transmission occurs when there is close contact with ill animals.20 To date, human-to-human transmission has not been observed.

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.

The availability of a rapidly expanding number of novel viral genomes identified by metagenomic studies has also presented challenges for virus taxonomy—a system for classification of viruses that is administered by the International Committee on Taxonomy of Viruses (ICTV). Should viruses detected only by their nucleotide sequence be classified and assigned to species and other higher taxa (genus, family order, etc.) alongside viruses for which we have a viable isolate? The ICTV (then the International Committee on Nomenclature of Viruses) was established in 1966 at the ninth International Congress for Microbiology in Moscow, publishing its first report in 1971. It operates under the auspices of the International Union of Microbiological Societies (IUMS), with the authority to develop, refine, and maintain a universal virus taxonomy. Historically, the description and classification of a new virus by the ICTV required significant information, such as host range, serology, replication cycles, and structure, aspects that could be determined from the study of isolates. On the other hand, sequences alone provide a trove of information, including evolutionary relationships (e.g., phylogeny), genome organization (e.g., the number of genes and their order), presence or absence of distinctive motifs (e.g., protein cleavage sites, terminal sequences, internal ribosome entry sites), as well as genome composition (e.g., codon usage, GC content), which of course could be used to inform classification into species. These concerns framed the contents of a workshop of experts and members of the ICTV, resulting in a seminal consensus statement in which viruses identified only from metagenomic data are considered to be bona fide viruses and thus candidates for taxonomic assignment. The expanding diversity of the known virosphere also presented challenges. A recent analysis of metagenomes of 3042 geographically and ecologically diverse samples led to the discovery of 125,842 new partial dsDNA viral genomes encoding more than 2.79 million proteins, 75% of which had no sequence similarity to proteins from known virus isolates. Other metagenomic studies have revealed similar diversity in ssDNA and RNA viruses, particularly in marine ecosystems. This has led ICTV to consider a far broader framework for taxonomic assignment of viruses, recently approving the establishment of a taxonomic hierarchy that includes 15 ranks (realm, subrealm, kingdom, subkingdom, phylum, subphylum, class, subclass, order, suborder, family, subfamily, genus, subgenus, and species), thus expanding the range even beyond those currently available for other organisms. The sheer volume of new virus genomes identified by metagenomic studies has also led to the development of new bioinformatic tools, that are increasingly being applied for automated virus classification of viruses, based almost exclusively on nucleotide sequence data.

Although no countries of the Eastern Mediterranean Region of WHO have reported importation of Zika virus disease or autochthonous transmission, however, of the 22 countries in the Region, the following 8 have reported dengue outbreaks in recent years and/or have the presence of competent Aedes mosquitoes: Djibouti, Egypt, Oman, Pakistan, Saudi Arabia, Somalia, Sudan and Yemen. Some of these countries are particularly vulnerable to emerging of Zika virus, because of the fragility of health systems, weakness of disease surveillance systems, the inadequacy of response capacities, and a suboptimal level of public health preparedness (29–31). Pakistan is the most-at-risk country in the Region, primarily due to a large number of travelers to and from the Americas, dense urban populations, a documented large epidemic of dengue, and the most favorable climatic conditions for the reproduction of Aedes mosquitoes (29). In Iran, national committee of Aedes-borne diseases recommended enhancement of surveillance after 2016 and updated national guidelines of surveillance and clinical management of suspected cases, especially for travelers returning from at-risk countries. As of Sep 2018, all of the samples from suspected cases of Zika virus sent to the National Laboratory of Aedes-borne diseases were shown to be negative. Prevention of Aedes-borne diseases needs comprehensive national strategic action plan with “one health” approach and close collaboration between community, academic, and public health authorities. Global map of Zika virus infection demonstrated in (Fig. 2) and areas potentially at risk of Zika in (Table 1).

Content source: Centers for Disease Control and Prevention, Sep 2018 updated. (

https://wwwnc.cdc.gov/travel/page/world-map-areas-with-zika

)

Depending on the purpose of the investigation, laboratory diagnosis of Zika virus can be conducted by virus isolation, antigen detection, viral RNA detection with molecular assays and anti-Zika virus antibodies detection with serological assays (35).

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).

The titles and abstracts of the identified studies were reviewed independently by two reviewers using Covidence Systematic Review software, (Veritas Health Innovation, Melbourne, Australia. Available at www.covidence.org). Differences and conflicts were resolved by a third reviewer and through discussions for a consensus to be reached. Percentage agreement and Cohen’s Kappa (κ) statistic were calculated and interpreted in accordance with Landis and Koch’s benchmarks for assessing the agreement between reviewers as poor (<0), slight (0.0–0.20), fair (0.21–0.40), moderate (0.41–0.60), substantial (0.61–0.80), and excellent (>0.81).

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.

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.

The genus flavivirus (family Flaviviridae) comprises several pathogens, including dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), tick-borne encephalitis virus (TBEV) and Japanese encephalitis virus (JEV). Flaviviruses that are pathogenic to man are transmitted to humans by bites of mosquitoes or ticks. The incidence and geographical distribution of the four distinct DENV serotypes and its vector are increasing dramatically. DENV causes more than 50 million infections annually (mainly in South-East Asia and Latin America) and infections with this virus may develop into dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS)–[4]. Increased disease severity has been associated with pre-existing heterologous DENV antibodies, a phenomenon described as antibody-dependent enhancement (ADE) of infection. Antibodies have been found to enhance viral entry into Fc-γ-receptor-bearing cells and to alter the antiviral immune response, leading to increased virus particle production and subsequent immune activation. During homologous re-infection, antibodies are believed to neutralize the infecting virus and provide life-long protection against disease development. Intriguingly, however, during heterologous re-infection, cross-reactive antibodies have been implicated to enhance viral replication leading to a higher infected cell mass and increased viral burden. There is neither a vaccine nor a specific antiviral therapy available. This is also the case for YFV that, together with DENV, is a leading cause of hemorrhagic fever worldwide, although a highly efficacious vaccine is available. Furthermore, vector-control strategies that were once successful in eliminating YFV have faltered, thereby leading to a re-emergence of the disease. The World Health Organization currently estimates that there are 200,000 cases of yellow fever annually of which over 90% occur in Africa, resulting in about 30,000 deaths per year. There is, as is the case for most of the other flaviviruses, no antiviral drug available for the treatment of YFV infections.

Glycopeptide antibiotics (i.e. teicoplanin, eremomycin and vancomycin) are used for the treatment of gram-positive bacterial infections. Synthetically modified glycopeptide antibiotics (SGPAs) have been reported to be endowed with in vitro antiviral activity against retro- and corona viruses,. For human immunodeficiency virus (HIV) it was shown that semisynthetic glycopeptide aglycons potentially interfere with the viral entry process. The mechanism by which deglycosylated SGPAs exert antiviral activity against other viruses remains unclear.

We recently demonstrated that the teicoplanin aglycon analogue LCTA-949 inhibits the replication of hepatitis C virus (HCV) by interfering with the intracellular replication of the virus. We here report that LCTA-949 also exerts in vitro anti-flavivirus activity and does so, surprisingly, by interfering with the very early stages of the viral replication cycle.

Infectious diseases have gained importance as a significant threat to public health following the recent outbreaks of arthropod-transmitted viruses (arboviruses) in the western hemisphere. Global emergence of arboviruses such as dengue virus (DENV), chikungunya, yellow fever virus (YFV), and Zika virus (ZIKV) has become possible due to several factors including urbanization, rapid population growth, and climate change. Although environmental changes have given importance to the movement of the human population, especially by air travel, the extent to which humans have reshaped the environment has also led to a dynamic spread of pathogens and their vectors. The dissemination of arboviruses, in particular, is dependent on their vectors, and reports of these pathogens in new global destinations should raise concerns of the expansive distribution of the vectors, such as the Aedes species of mosquitoes. Of the numerous human pathogens that have experienced a rapid geographic distribution, this review focuses on ZIKV, which has raised international health concerns due to its broad spectrum of transmission routes, autoimmune disorders in adults, and neurodevelopmental complications in newborns. ZIKV belongs to the Flaviviridae family along with Japanese encephalitis virus (JEV), West Nile virus (WNV), and DENV, all of which are medically important viruses transmitted by mosquitoes or ticks. As of January 2018, PAHO has reported 223,477 confirmed ZIKV cases that have cumulated worldwide between 2015 and 2018. Despite the global distribution of ZIKV, there are no clinically approved vaccines or therapeutic treatments available to combat the infections. As a result, international concern surrounding ZIKV in terms of control, treatment, and prevention has classified the virus as a global threat to public health.

Although most cases of ZIKV infection result in asymptomatic or mild flu-like symptoms, such as fever, rash, and conjunctivitis, the series of outbreaks that started in Yap islands in 2007 has emphasized just how wide of a phenotypic spectrum of disease can be caused in humans by the virus. Recent incidences of infection have resulted in severe phenotypes including Guillain–Barré syndrome, meningoencephalitis, and fetal abnormalities such as microcephaly and spontaneous abortion. Up to date, diagnosis of ZIKV infection has depended on molecular and serological testing, employing ELISA and RT-PCR platforms for IgM and RNA detection accordingly. However, these methods of diagnosis are only useful for virus detection within a short frame from the symptom onset as levels of viral RNA and IgM antibodies decline over time. Laboratory testing of infants suspected of congenital ZIKV infection includes detection of viral RNA in serum and urine, and IgM antibodies in serum and CSF of infants. Although tests should be performed as early as possible once an infection is suspected, the optimal timing and type of specimen and assay for detection remain undetermined.

Since the series of outbreaks in 2007, ZIKV infection has increasingly been associated with cases of neurological complications and congenital malformations. Microcephaly, which is defined by a brain size that is at least two standard deviations below the mean, is one clinical presentation of a congenital ZIKV infection, and the World Health Organization has already established an etiological link between ZIKV infection and birth defects like microcephaly in Brazil. Congenital abnormalities induced by ZIKV infection have confirmed the possibility of vertical transmission. Despite the prevalence of birth defects, diagnosis of congenital microcephaly remains a challenge due to the existence of various etiological factors involved. Pathogenesis studies have confirmed the neurotropism of ZIKV, although the exact molecular mechanism of neuropathogenesis remains unclear. In addition to microcephaly, exposure to ZIKV during pregnancy can result in visual and hearing impairments in the newborn. Thus, the phenotypic spectrum of outcomes of pregnancy-associated ZIKV infection has identified ZIKV as a dangerous and atypical member among the flaviviruses.

Flaviviruses carry out their life cycle by utilizing machinery and functions of the host cell. Consequently, flavivirus–host cell interactions are essential for the pathogenesis. Many of these crucial interactions, however, remain elusive. In this Review, we focus on ZIKV, a member of the Flaviviridae family, and highlight the viral pathogenesis at the level of cellular mechanisms and interactions.

In the twenty-first century, we have seen the (re-)emergence of several RNA viruses causing severe infections in humans, like SARS and MERS coronavirus, and more recently Ebola virus and Zika virus (ZIKV). A problem with (re-)emerging virus infections is the lack of available medical countermeasures, including antiviral treatment. Therefore, the development of broadly acting antiviral compounds, as well as screening of existing drugs for potential repurposing are explored as ways to fast-track the drug development pipeline for emerging viral diseases. Here, we briefly discuss the available information on potential antiviral treatment of ZIKV infection, as well as specific challenges regarding use of antivirals.

Zika virus is a positive-sense single-stranded RNA arbovirus that belongs to the genus Flavivirus of the family Flaviviridae. Based on nucleotide sequences, ZIKV can be further divided into an African lineage and an Asian lineage. The main vector for ZIKV transmission are Aedes mosquitoes. The first isolation of ZIKV was made in 1947 from serum of a febrile rhesus monkey in the Zika forest of Uganda. During the twentieth century, sporadic human cases of ZIKV infection were reported in Africa caused by the African lineage, and in Asia caused by Asian lineage. The epidemiology of ZIKV changed dramatically in the last decade, when major outbreaks of the Asian lineage of ZIKV were reported on the Island of Yap in 2007 and in French Polynesia in 2013. Subsequently, the Asian lineage of ZIKV spread to the Americas in 2015.

The current outbreak of ZIKV shows new modes of transmission with mother-to-fetus transmission and sexual transmission. Furthermore, infectious ZIKV has been detected in breast milk, and there are concerns of ZIKV transmission by blood products obtained from asymptomatic viremic persons or by organ transplantation.

While postnatally acquired ZIKV infections are asymptomatic or associated with mild symptoms, the recent outbreaks brought two severe complications to light: a Guillain-Barre-like syndrome (GBS), associated with paralysis, and microcephaly and other neuro-developmental problems in infants born to women infected with ZIKV during pregnancy. The rate of complications has been estimated to be around 1/4000 cases for the GBS-like syndrome, and may be considerably higher for the teratogenic effects, although much remains unknown about the precise extent of the possible sequelae in fetuses and infants. A significant contributing factor to this knowledge gap is that up to 80 % of Zika virus infections may be asymptomatic, and these ‘silently’ infected women with potentially affected fetuses and babies may go unnoticed.

There are currently no drugs approved for treatment, but several nucleoside analogue drugs have some antiviral activity in cell culture such as 2′-C-methylated nucleosides like 7-deaza-2′-C-methyladenosine (7DMA) and 2′-C-methylcytidine (2CMC), and ribavirin, favipiravir, and T-1105 (Fig. 1). In addition, 7DMA shows modest anti-ZIKV activity in mice. Ribavirin and favipiravir are already approved for use as antiviral drugs in humans for other indications, but showed less in vitro antiviral effect against ZIKV than 7DMA. 7DMA and 2CMC were initially developed for treatment of hepatitis C virus (HCV) which is distantly related to ZIKV, and studies have shown that these compounds have activity against other flaviviruses much more closely related to ZIKV like dengue virus (DENV). Of interest, other anti-HCV nucleoside analogues like the approved sofosbuvir and mericitabine (in clinical trials) also showed in vitro activity against DENV. GS-5734 and BCX4430 are two very broad spectrum anti-RNA virus nucleoside analogues that are currently in phase 1 and 2 clinical trials. According to the manufacturer, BCX4430 reduced mortality in ZIKV-infected mice, but this data has yet to be published.

The above shows that there is some evidence for the use of broad spectrum antiviral drugs against ZIKV, in particular broad spectrum nucleoside analogues, but the level of evidence thus far is insufficient for licensed clinical applications. Also, a major challenge is how and when such antiviral treatment would be applied, if available. The drug should be active in cells of the nervous system; it should cross the blood-brain-barrier and the placenta, and should be safe for pregnant women, their fetuses, and infants, which are by no means trivial concerns. For example, the unfavorable toxicity profile of ribavirin including teratogenicity limits its use to treat ZIKV infections in pregnant women. In addition, a lesson learned from treating other RNA viruses is that monotherapy can cause the rapid emergence of resistant viruses, stressing the need for development of drugs with different modes of action to reduce the likelihood of selecting out resistant strains.

Given the time needed to develop and approve new antiviral drugs, clinicians are currently left empty handed in terms of targeted antiviral treatment for ZIKV infection. A major lesson from this outbreak and previous (re-)emerging RNA virus outbreaks is that we need safe, broad spectrum antiviral drugs that can be tested rapidly in phase III clinical trials during outbreaks to gain the time needed for the development of other control and prevention measures, particularly vaccines.

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.

The genus Flavivirus belongs to the Flaviviridae family and includes more than 70 single-stranded plus-sense RNA viral species. Flaviviruses of human medical importance are tick- or mosquito-transmitted viruses with typical representatives being tick-borne encephalitis virus (TBEV), Omsk hemorrhagic fever virus (OHFV), Kyasanur Forest disease virus (KFDV), Alkhurma hemorrhagic fever virus (AHFV), Powassan virus (POWV), West Nile virus (WNV), dengue virus (DENV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), or Zika virus (ZIKV).1,2 The Flaviviridae family also includes some less known or neglected viruses, such as louping ill virus (LIV), Usutu virus, Langat virus, or Wesselsbron virus.3–6 The flaviviral genome is a single-stranded, plus-sense RNA of about 11 kb in length that encodes a single polyprotein, which is co- and posttranslationally processed into three structural (capsid, premembrane or membrane, and envelope) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).7 Both NS3 and NS5 proteins possess enzymatic activities reported to be important targets for antiviral development. Whereas NS3 acts as a serine protease, a 5′-RNA triphosphatase, a nucleoside triphosphatase (NTPase), and a helicase,8,9 NS5 consists of a complex containing the RNA-dependent RNA polymerase (RdRp) and the methyltransferase (MTase) activities.10,11

Flaviviral infections are accompanied by a wide spectrum of distinct clinical manifestations, ranging from relatively mild fevers and arthralgia to severe viscerotropic symptoms (YFV and DENV), hemorrhagic fevers (KFDV and OHFV), encephalitis/myelitis (JEV, WNV, and TBEV), and neuropathic or teratogenic manifestations (ZIKV). More than 200 million clinical cases of flaviviral infections, including numerous deaths, are reported annually worldwide.12 Currently no specific antiviral therapies are available to treat patients with flaviviral infections, thus the search for safe and effective small-molecule inhibitors that would be active against these viruses represents a high research priority.13

Nucleoside analog inhibitors have figured prominently in the search for effective antiviral agents.14 Nucleoside analogs are synthetic, chemically modified nucleosides that mimic their physiological counterparts (endogenous nucleosides) and block cellular division or viral replication by impairment DNA/RNA synthesis or by inhibition of cellular or viral enzymes involved in nucleoside/tide metabolism (Figure 1).15 The first antiviral analogs were developed in the late 1960s and currently there are over 25 approved therapeutic nucleosides used for the therapy of viral infections of high medical importance, such as HIV/AIDS (tenofovir),16,17 hepatitis B (lamivudine/entecavir),18,19 hepatitis C (sofosbuvir),20 or herpes infections (acyclovir).21 So far, numerous nucleoside analogs have been described to inhibit arthropod-transmitted flaviviruses. Since these viruses are closely related to the hepatitis C virus (HCV), for which many potent inhibitors are being currently developed, anti-HCV nucleoside analogs represent promising tools to be repurposed against other viruses within the Flaviviridae family.12

The aim of this review is to provide an overview of known antiviral agents targeting selected arthropod-borne flaviviruses and to discuss their characteristic properties, modes of action, and advantages or limitations of their therapeutic use. Moreover, the important challenges and complications in antiflavivirus nucleoside analog development are highlighted and possible strategies to overcome their shortcomings are suggested.

Zika virus (ZIKV) belongs to the genus Flavivirus within the Flaviviridae family. ZIKV is an enveloped positive sense single-stranded RNA virus with a genome size of ∼10.7 kb that encodes a single polyprotein, which is post-translationally processed by cellular and viral proteases into three structural (capsid, C; pre-membrane, prM; and envelope, E) and seven non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins (Tripathi et al., 2017; Avila-Perez et al., 2018).

Zika virus was initially isolated from Uganda in 1947 and viral infections only occurred sporadically in Africa and Asia until 2007. ZIKV appeared explosively as the first large-scale outbreak occurred in the Yap island in 2007 and French Polynesia in 2013 (Weaver et al., 2016). Most recently, in 2015, the first local transmission of ZIKV was found in territories of Latin America and the Caribbean, resulting in up to 1.3 million of ZIKV infection suspected cases (Tang et al., 2016; Tripathi et al., 2017).

Like other members of the Flaviviridae family, such as yellow fever virus (YFV), Dengue virus (DENV), Japanese encephalitis virus (JEV), and West Nile virus (WNV), ZIKV is commonly transmitted by the bite of infected Aedes mosquitos, but it can also be transmitted vertically from mother to child, through sexual contact, and in rare cases from blood transfusions (Lessler et al., 2016; Fink et al., 2018). Upon infection, ZIKV can be shed in blood, urine, semen, saliva, amniotic fluid, breast milk, and cerebrospinal fluid (Nayak et al., 2016; Colt et al., 2017; Nazerai et al., 2019). Most people (75∼80%) infected with ZIKV are asymptomatic or have mild symptoms such as fever, rash, joint pain, and conjunctivitis that can last for several days to a week (Fink et al., 2018). In rare cases, people with symptoms may have neurological Guillain-Barré syndrome complications (Oehler et al., 2014; Rivera-Concepcion et al., 2018; Nazerai et al., 2019). In the case of pregnant women, ZIKV infection can lead to microcephaly and other fetal complications as occurred during the large-scale ZIKV outbreak in Brazil in 2015 (Lessler et al., 2016). Because of the significant outbreaks in South, Central, and North America, ZIKV was declared a Public Health concern by the World Health Organization (WHO) in February 2016 (Lazear and Diamond, 2016; Ramos da Silva and Gao, 2016; Weaver et al., 2016; Tripathi et al., 2017).

There are several vaccines and antiviral drugs currently under development for the prevention or treatment of ZIKV infection (Abbink et al., 2016; Larocca et al., 2016; Shan et al., 2017; Fink et al., 2018). DNA-based (Abbink et al., 2016; Larocca et al., 2016), inactivated (Abbink et al., 2016; Larocca et al., 2016; Shan et al., 2017), live-attenuated and mRNA (Richner et al., 2017) vaccines have been proposed for the prophylactic treatment of ZIKV infections. On the other hand, arbidol (ARB) (Fink et al., 2018; Haviernik et al., 2018), bortezomib, mycophenolic acid, daptomycin (Barrows et al., 2016), obatoclax, saliphenylhalamide, gemcitabine (Kuivanen et al., 2017), emetine (Yang et al., 2018), and sofosbuvir (Bullard-Feibelman et al., 2017) have been proposed for the therapeutic treatment of ZIKV infection. Despite these tremendous efforts, there is currently no Food and Drug Administration (FDA)-approved vaccines and/or anti-viral drugs available for the treatment of ZIKV infection. Since vaccination takes at least 2 weeks to several months to show protective effects against ZIKV infection, vaccination is probably not the most appropriate prophylactic method for those who are traveling to areas where ZIKV is epidemic, endemic, or have already been infected. Moreover, vaccination may cause an important issue, such as antibody-dependent enhancement (ADE) (Bardina et al., 2017; Priyamvada et al., 2017). ADE, which has been extensively described in DENV (Priyamvada et al., 2017), is a phenomenon where preexisting antibodies facilitate binding and infection during subsequent exposure to infectious viruses, instead of neutralizing them, resulting in exacerbation of clinical signs (Bardina et al., 2017; Priyamvada et al., 2017). Because of the structural similarities between DENV and ZIKV, DENV immunity–linked ADE of ZIKV infection has also been reported (Bardina et al., 2017; Priyamvada et al., 2017). Since vaccination for ZIKV could lead to DENV ADE, antivirals could represent a better choice for the control of ZIKV infection.

Aurintricarboxylic acid (ATA), a polyanionic aromatic compound, has been shown to have inhibitory properties against several bacteria and viruses including, among others, Yersinia pestis (Liang et al., 2003), Cryptosporidium parvum (Klein et al., 2008), human immunodeficient virus (HIV) (Mitra et al., 1996; De Clercq, 2005), hepatitis C virus (HCV) (Chen et al., 2009; Mukherjee et al., 2012; Shadrick et al., 2013), Vaccinia virus (Myskiw et al., 2007), influenza virus (Hung et al., 2009), Enterovirus 71 (Hung et al., 2010) and severe acute respiratory syndrome coronaviruses (SARS-CoV) (He et al., 2004). Mechanistic studies have suggested that ATA has the ability to modulate various cellular enzymes such as activators of the Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5) families (Rui et al., 1998), inhibitors of nucleases (Shadrick et al., 2013), glucose-6-phosphate dehydrogenase (Bina-Stein and Tritton, 1976), and topoisomerase II proteins (Catchpoole and Stewart, 1994; Benchokroun et al., 1995) as well as the enzymatic activity of the Vaccinia virus AH1L phosphatase (Smee et al., 2010). However, to date, the ability of ATA to inhibit ZIKV infection has not been evaluated. Herein, we investigated ATA as a plausible prophylactic and therapeutic candidate against ZIKV infection. Our results demonstrate that ATA has a potent and effective antiviral activity against ZIKV in pre- and post-infection settings, including broadly antiviral activity against strains of the African and American/Asian lineages with no toxicity up to 1,000 μM in cultured cells. These data support the feasibility of implementing ATA for the treatment of ZIKV infection.

The possible cross amplification of other vesicular etiological agents was examined by using vesicular diagnostic specimens from bovine, a species not considered susceptible to SV-A infection. A total of 37 archived bovine tissues were analyzed (including vesicular fluid, swabs and epithelium from hoof lesions, epithelium, nasal swab, oral probang sample, etc.), from 33 animals with signs of vesicular disease, and found to be negative for high consequence agents such as FMDV and VSV, although some of them were positively diagnosed for the presence of other viruses such as bluetongue virus (BTV), epizootic hemorrhagic disease virus (EHDV), and bovine popular stomatitis virus; strains found in the US domestic animal population (data not shown). These specimens were all found to be negative for SV-A RNA with this assay, indicating that the RT-qPCR primers selected in this study do not cross-react with common viruses responsible for look-a-like vesicular diseases in cattle.

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].

Detection of SV-A antibodies in field animals with vesicular lesions has been attributed to possible association of the virus with SIVD syndrome [4, 5]. Differential diagnosis of SIVD from high consequence vesicular animal diseases, such as FMD, is crucial due to trade and movement restriction and other economic impacts associated with foreign animal diseases. Therefore, FADDL routinely investigates the presence of SV-A as the possible cause of vesicular lesions in swine while performing priority diagnosis from other devastating vesicular diseases including FMD by respective disease-specific RT-qPCR assays. The SV-A RT-qPCR test developed in this study complemented our laboratory needs for a fast, specific, sensitive and quantitative assay. Once demonstrated as sensitive, specific and reproducible in feasibility studies, we applied it to analyze swine vesicular diagnostic specimens that were previously found negative of the foreign animal disease agents FMD, VS, SVD and VES. Surprisingly, 88% of animals with vesicular lesions were positive for SV-A by RT-qPCR, confirming the previous observation that the virus appears to be associated with SIVD. The SV-A conventional PCR was previously performed upon request only, and the SVV qRT-PCR assay was only performed regularly starting in June 2015 on all swine vesicular investigation submissions, due to the increased prevalence of the virus. The virus was detected in several types of tissue specimens suggesting that it is ubiquitously distributed in different organs of an infected animal. Some of the RNA templates demonstrated very low Ct values (<14) suggesting a high presence of SV-A particles in those specimens. Additionally, the virus could not be detected in any of the tissues from the 35 pigs that did not possess SIVD signature clinical signs.

Previously, the presence of SV-A neutralizing antibody was found in sera from pigs irrespective of the disease status [http://www.europic.org.uk/Europic2006/posters/Knowles.svv.01.pdf]. In this study, we were able to directly detect the low levels of SV-A RNA, presumably viral genome, in 27% of sera from swine with or without SIVD symptoms. This included 3 feral swine indicating that wildlife can serve as the possible natural reservoir of the virus. Recently, Yang et al 2012 described the development of a SV-A indirect and cELISA assays which could be used for specific detection of SV-A serum antibodies. While farm animals are routinely monitored for appearance of vesicular lesions and precautionary laboratory diagnoses are done on suspect animals, feral swine are less frequently observed for vesicular disease. Pathogenicity of the virus remains unknown, and a cause for the observed high frequency of SV-A infection in animals presenting with vesicular disease is not yet understood. It is likely that environmental stressors or other infectious agents may be required in addition to SV-A for induction of SIVD. This assumption is supported by our frequent detection of other porcine viruses along with SV-A in clinical field animals and on an observed correlation from case histories indicating stressful conditions, such as movement, and the presence of SV-A in vesicular tissues presented in Table 2. An additional factor complicating the understanding of pathogenicity is the potential for sequence variation between strains of SV-A. Employing sensitive and specific detection methods for SV-A in rule-out diagnostic investigations of high consequence vesicular diseases in swine may not only help to resolve such disease investigations but ultimately may help to better understand the factors contributing to the presentation of SIVD.