Despite the announcement of the successful eradication of smallpox in 1979, the last case of rinderpest in 2008 and the current campaigns to eradicate poliomyelitis and measles through mass-immunization programmes, we still face the prospect of emerging or reemerging viral pathogens that exploit changing anthropological behavioural patterns. These include intravenous drug abuse, unregulated marketing of domestic and wild animals, expanding human population densities, increasing human mobility, and dispersion of livestock, arthropods and commercial goods via expanding transportation systems. Consequently, the World Health Organization concluded that acquired immune deficiency syndrome, tuberculosis, malaria, and neglected tropical diseases will remain challenges for the foreseeable future.1 Understandably, the high human fatality rates reported during the recent epidemics of Ebola, severe acute respiratory syndrome and Middle East respiratory syndrome have attracted high levels of publicity. However, many other RNA viruses have emerged or reemerged and dispersed globally despite being considered to be neglected diseases.2,3 Chikungunya virus (CHIKV), West Nile virus (WNV) and dengue virus (DENV) are three of a large number of neglected human pathogenic arthropod-borne viruses (arboviruses) whose combined figures for morbidity and mortality far exceed those for Ebola, severe acute respiratory syndrome and Middle East respiratory syndrome viruses. For instance, for DENV, the number of cases of dengue fever/hemorrhagic fever is between 300–400 million annually, of which an estimated 22 000 humans die.4 Moreover, in the New World, within 12 months of its introduction, CHIKV caused more than a million cases of chikungunya fever according to Pan American Health Organization/World Health Organization, with sequelae that include persistent arthralgia, rheumatoid arthritis and lifelong chronic pain.5 Likewise, within two months of its introduction, to Polynesia, the number of reported cases exceeded 40 0006 and is currently believed to be approaching 200000 cases. Alarmingly, this rapid dispersion and epidemicity of CHIKV (and DENV or Zika virus in Oceania) is now threatening Europe and parts of Asia through infected individuals returning from these newly endemic regions. This is an increasingly worrying trend. For example, in France, from 1 May to 30 November, 2014, 1492 suspected cases of dengue or chikungunya fever were reported.7 Accordingly, this review focuses on the emergence or reemergence of arboviruses and their requirements and limitations for controlling these viruses 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.

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.

Alpaca (Vicugna pacos, also known as Lama guanicoe pacos) are domesticated members of the New World camelid species (Lamini), which also include guanaco (Lama guanicoe), vicuna (Vicugna vicugna), and llama (Lama glama). The natural habitat for alpaca is at high altitude (3500–5000 m) in South America (Peru, Ecuador, Bolivia, and Chile) where they are kept as livestock in herds and their fiber is used much like wool. Approximately 300,000 animals are in the U.S. Compared to other livestock, e.g., about 96 million cattle, their number is still relatively small.

Previously reported viral infections in domestic alpaca include adenovirus, equine viral arteritis virus, rabies, bluetongue virus, foot-and-mouth disease virus, bovine respiratory syncytial virus, influenza A virus, rotavirus, orf virus, bovine papillomavirus, vesicular stomatitis virus, coronavirus, bovine parainfluenza-3 virus, West Nile virus, equine herpesvirus-1,, and bovine viral diarrhea virus–[13]. Bovine enteroviruses (BEV) have not previously been reported to infect alpaca. The bovine enterovirus species previously contained two types, BEV-A and BEV-B, although a new classification structure was ratified recently, redesignating these as species Enterovirus E (EV-E) and Enterovirus F (EV-F), respectively,. Each of the new BEV species includes multiple serotypes, with EV-E comprising four described serotypes (previously A1–4, renamed E1–E4), and EV-F containing six reported serotypes (previously B1–6, renamed F1–F6).

Recently developed approaches to virus detection have the potential to further expand understanding of viral disease in animals, including alpaca. Many of these approaches are based on non-specific PCR amplification used in conjunction with standard or high-throughput sequencing to identify PCR products.

We utilized such a method–[19] to investigate an outbreak of a respiratory infection in alpaca, identifying a bovine enterovirus (EV-F), named Enterovirus F, strain IL/Alpaca, after other techniques had failed to detect any pathogen.

Arboviruses are arthropod-borne viruses that exhibit worldwide distribution and are a constant threat, not only for the public health but also for wildlife, domestic animals, and even plants. The rise in global travel and trade as well as the changes in the global climate conditions are facilitating the expansion of the vector transmitters, including mosquitoes, ticks, sandflies, and midges among other arthropods, from endemic to new areas, augmenting the number of outbreaks around the world at an unprecedented rate. Arboviruses need multiple hosts to complete their cycle (i.e., host and vector), making it possible to impact disease by targeting either the arthropod vector and/or the pathogen. For some of these pathogens, efficient antivirals or vaccines are not available, in some cases due to the genetic variability of these viruses. Moreover, there are a limited availability of animal models to study infections, and some of them display a poor immunogenicity and some others viral infections cause neglected diseases that have not been deeply studied. Transmission between the vector and the host occurs when the vector feeds on the blood of the host by biting. However, the vector does not act as a simple vehicle that passively transfer viruses from one individual to another. Instead, arthropod-derived factors found in their saliva have an important role in infection and disease, modulating (positively and negatively) replication and dissemination within the host. In addition, the inflammatory response that the host mounts against these vector molecules can enhance the severity of arbovirus infection.

To study disease pathogenesis and to develop efficient and safe therapies to prevent (vaccines) or treat (antivirals) viral infections, the use of an appropriate animal model is a critical concern. The use of mice as small animal models to study immunity, pathogenesis, as well as to test candidate vaccines and antivirals against a largely variety of viral diseases is widely spread. They are cost effective, being affordable for most of research laboratories. They reproduce quickly, are easy to handle, do not require specialized facilities to house, and multiple inbred strains of genetically identical mice are available. In many cases such as Crimean Congo Hemorrhagic Fever (CCHFV), Bluetongue (BTV), Middle East respiratory syndrome (MERS), or Ebola (EBoV) viruses, the pathogenesis of disease in humans is also partially mimicked. Furthermore, optimal reagents have been developed for in vivo and in vitro studies in mice, a fact which allows the study of other animal viruses apart from those which are human specific. Also, it is possible to manipulate the mouse genome and generate transgenic, knock-out, knock-in, humanized, and conditionally mutant strains to interrogate protein function in physiological and pathological signs.

Immunocompetent wild-type mice are susceptible to infections with a number of viral pathogens such as influenza virus; severe acute respiratory syndrome coronavirus (SARS-CoV); and Rift Valley fever virus (RVFV). Unfortunately, immunocompetent mice are not susceptible to many other viruses with outbreak potential, and thus alternative strategies are needed.

West Nile virus is the etiological agent of an emerging zoonotic disease whose impact on animal and public health is considerable, being the most widespread arbovirus in the world today (reviewed in Hayes et al., 2005a; Kramer et al., 2008; Brault, 2009). A percentage of WNV infections result in severe encephalitis, and it is a communicable disease both for human and animal health. WNV taxonomically belongs to the family Flaviviridae, genus Flavivirus. Virions are spherical in shape, about 50 nm in diameter, and consist of a lipid bilayer that surrounds a nucleocapsid that in turn encloses the genome, a unique single-stranded RNA molecule, which encodes a polyprotein that is processed to give the 10 viral proteins. Of them, three (C, E, and M) form part of the structure of the virion, and the rest (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5) are so-called “non-structural” and play important roles in the intracellular processes of replication, morphogenesis, and virus assembly. Inserted into the lipid bilayer are two proteins, E (from “envelope”) and M (“matrix”), which participate in important biological properties of the virus, such as its host range, tissue tropism, replication, assembly, and stimulation of cellular and humoral immune responses. E protein contains the major antigenic determinants of the virus.

As far as we know, there are no serotypes of WNV, but two main genetic variants or lineages can be distinguished, namely lineages 1 and 2. While the former is widely distributed in Europe, Africa, America, Asia, and Oceania, the second is found mostly restricted to Africa and Madagascar, although it has recently been introduced in Central and Eastern Europe (Bakonyi et al., 2006; Platonov et al., 2008) and has further extended to southern Europe (Bagnarelli et al., 2011; Papa et al., 2011). In addition, other viral variants closely related phylogenetically to WNV have been described, which are different from lineages 1 and 2, and have been proposed as additional WNV lineages. One of them, known as “Rabensburg virus,” isolated form mosquitoes in the Czech Republic in 1997, shows low pathogenicity in mice (Bakonyi et al., 2005). Similarly, other viruses closely related to WNV have been isolated in India (Bondre et al., 2007), Russia (Lvov et al., 2004) Malaysia (Scherret et al., 2001), and Spain (Vazquez et al., 2010). All these viruses have been proposed to represent different genetic lineages of WNV. Except for the Indian variant, which has been involved in outbreaks of encephalitis in humans, the rest are of unknown relevance for animal and human health.

West Nile fever/encephalitis is a disease transmitted mainly by mosquitoes, while wild birds are its natural reservoir. WNV is capable of infecting a wide range of bird species. Nevertheless, birds were considered less susceptible to the disease until the recent epidemic of WNV in North America, affecting many species of birds lethally, made to re-examine this concept (Komar et al., 2003). Occasionally it may affect poultry species, mainly geese and ostriches. Other domestic birds like chickens and pigeons, are susceptible to infection but do not get sick, and are often used as sentinels for disease surveillance. In addition to birds, WNV can also affect a wide range of vertebrates species, including amphibians, reptiles, and mammals, and it is particularly pathogenic in humans and horses, which act epidemiologically as “dead end hosts,” that is, they are susceptible to infection but do not transmit the virus (McLean et al., 2002; Kramer et al., 2008).

The first case of WNF was described in Uganda (West Nile district, hence the name of the virus) in a feverish woman, from whose blood the virus was first isolated in 1937 (Smithburn et al., 1940). It was considered a mild disease, endemic in parts of Africa (an “African fever”). However, since around 1950s, the occurrence of disease outbreaks with neurological disease, lethal in some cases, caused by WNV, especially in the Middle East and North Africa, made necessary to rethink this concept. In humans, the majority of WNV infections are asymptomatic, about 20% may develop mild symptoms such as headache, fever, and muscle pain, and less than 1% develop more severe disease, characterized by neurological symptoms, including encephalitis, meningitis, flaccid paralysis, and occasionally severe muscle weakness (Hayes et al., 2005b). Advanced age is considered a risk factor for developing severe WNV infection or death. The mortality rate calculated for the recent epidemic of the disease in the U.S. is 1 in every 24 human cases diagnosed (Kramer et al., 2008).

In horses (reviewed in Castillo-Olivares and Wood, 2004) neurological disease is manifested by approximately 10% of infections, and is mainly characterized by muscle weakness, ataxia, paresis, and paralysis of the limbs, as a result of nerve damage in the spinal cord. They may also suffer from fever and anorexia, tremors and muscle stiffness, facial nerve palsy, paresis of the tongue, and dysphagia, as a result of affection of the cranial nerves. A proportion of horses infected with WNV die spontaneously or is slaughtered to avoid excessive suffering. The mortality rate can vary between outbreaks. For example, in the outbreak in 2000 in the Camargue (France), 76 horses were affected, of which 21 died (Zeller and Schuffenecker, 2004). In 1996 in Morocco, a WNV outbreak affected 94 horses, of which 42 died (Zeller and Schuffenecker, 2004). Severe equine cases do not seem to predominate in older horses, as occurs in humans (Castillo-Olivares and Wood, 2004). Other mammals may also suffer from the disease. Rodents such as laboratory mice and hamsters are highly susceptible, so they can be used as experimental model of WNV encephalitis. Lemurs and certain types of squirrels appear to be the only mammals capable of maintaining the virus in local circulation (Rodhain et al., 1985; Root et al., 2006). WNV can also infect other mammals, including sheep, in which it causes abortions, but rarely encephalitis (Hubalek and Halouzka, 1999). WNV has been isolated from camels, cows, and dogs in enzootic foci (Hubalek and Halouzka, 1999). The virus has been shown to infect frogs (Rana ridibunda), which in turn are bitten by mosquitoes, so that the existence of an enzootic cycle in these amphibians is postulated, at least for some variants of the virus (Kostiukov et al., 1986). Outbreaks of severe WNF with high mortality have been reported in captive alligators and crocodiles, presumably transmitted through feeding of contaminated meat (Miller et al., 2003). It has been shown experimentally that WNV can infect asymptomatically pigs (Teehee et al., 2005) and dogs (Blackburn et al., 1989; Austgen et al., 2004). However, guinea pigs, rabbits, and adult rats are resistant to infection with WNV (McLean et al., 2002). Among non-human primates, rhesus and bonnet monkeys (but not Cynomolgus macaques and chimpanzees), inoculated with WNV develop fever, ataxia, prostration with occasional encephalitis and tremor in the limbs, paresis or paralysis. The infection can be fatal in these animals.

The virus is propagated in the reservoir hosts, resulting in a viremic phase that usually lasts no more than 5–7 days (Komar et al., 2003). The duration and level of viremia depends on the species infected (Komar et al., 2003). The detection of the virus or its genetic material in serum or cerebrospinal fluid in a laboratory test is a proof of diagnostic value (De Filette et al., 2012). The virus is evidenced by virological (virus isolation) or molecular (RT-PCR-conventional and real-time, NASBA) techniques. In epidemiological surveillance it is useful to detect the presence of WNV in mosquitoes, for which they are homogenized and analyzed using the same methods mentioned above (Trevejo and Eidson, 2008). Specific antibodies against the virus are detectable in blood few days after infection (Komar et al., 2003; De Filette et al., 2012). Antibody detection is performed by serological tests (enzyme immunoassay or ELISA, hemagglutination inhibition or HIT) which can be confirmed by more specific serological techniques (virus-neutralization test; Sotelo et al., 2011c). Serological diagnosis of acute infection should be done by detection of IgM antibodies in serum and/or cerebrospinal fluid using an immunocapture ELISA together with the detection of an increase in antibody titer in paired sera taken one in the acute phase and the other, at least 2 weeks later (Beaty et al., 1989).

The fight against this disease is not straightforward because there are no vaccines licensed for human use, and even though there are some available for veterinary use, they are efficacious to prevent disease symptoms and outcome at the individual level but do not prevent the spread of the infection, mainly due to the establishment of an enzootic cycle among wild birds and mosquitoes (Kramer et al., 2008; De Filette et al., 2012). Control methods are mainly based on prevention and early detection of virus spread through epidemiological surveillance and targeted application of insecticides and larvicides (Kramer et al., 2008).

Depending upon the involvement of etiological agent, the infectious respiratory diseases of small ruminants can be categorized as follows [9, 14]:bacterial: Pasteurellosis, Ovine progressive pneumonia, mycoplasmosis, enzootic pneumonia, and caseous lymphadenitis,viral: PPR, parainfluenza, caprine arthritis encephalitis virus, and bluetongue,fungal: fungal pneumonia,parasitic: nasal myiasis and verminous pneumonia,others: enzootic nasal tumors and ovine pulmonary adenomatosis (Jaagsiekte).

Manytimes due to environmental stress, immunosuppression, and deficient managemental practices, secondary invaders more severely affect the diseased individuals; moreover, mixed infections with multiple aetiology are also common phenomena [5, 8, 13, 15].

These conditions involve respiratory tract as primary target and lesions remain confined to either upper or lower respiratory tract [7, 16]. Thus, these diseases can be grouped as follows [5, 8, 14, 17].Diseases of upper respiratory tract, namely, nasal myiasis and enzootic nasal tumors, mainly remain confined to sinus, nostrils, and nasal cavity. Various tumors like nasal polyps (adenopapillomas), squamous cell carcinomas, adenocarcinomas, lymphosarcomas, and adenomas are common in upper respiratory tracts of sheep and goats. However, the incidence rate is very low and only sporadic cases are reported.Diseases of lower respiratory tract, namely, PPR, parainfluenza, Pasteurellosis, Ovine progressive pneumonia, mycoplasmosis, caprine arthritis encephalitis virus, caseous lymphadenitis, verminous pneumonia, and many others which involve lungs and lesions, are observed in alveoli and bronchioles.

Depending upon the severity of the diseases and physical status of the infected animals, high morbidity and mortality can be recorded in animals of all age groups. These diseases alone or in combination with other associated conditions may have acute or chronic onset and are a significant cause of losses to the sheep industry [3, 10]. Thus, the respiratory diseases can also be classified on the basis of onset and duration of disease as mentioned below [3, 9, 14, 18]:acute: bluetongue, PPR, Pasteurellosis, and parainfluenza,chronic: mycoplasmosis, verminous pneumonia, nasal myiasis, and enzootic nasal tumors,progressive: Ovine progressive pneumonia, caprine arthritis encephalitis virus, caseous lymphadenitis, and pulmonary adenomatosis.

Small ruminants particularly sheep and goats contribute significantly to the economy of farmers in Mediterranean as well as African and Southeast Asian countries. These small ruminants are valuable assets because of their significant contribution to meat, milk, and wool production, and potential to replicate and grow rapidly. The great Indian leader and freedom fighter M. K. Gandhi “father of the nation” designated goats as “poor man's cow,” emphasizing the importance of small ruminants in poor countries. In India, sheep and goats play a vital role in the economy of poor, deprived, backward classes, and landless labours. To make this small ruminant based economy viable and sustainable, development of techniques for early and accurate diagnosis holds prime importance. Respiratory diseases of small ruminants are multifactorial and there are multiple etiological agents responsible for the respiratory disease complex. Out of them, bacterial diseases have drawn attention due to variable clinical manifestations, severity of diseases, and reemergence of strains resistant to a number of chemotherapeutic agents. However, sheep and goat suffer from numerous viral diseases, namely, foot-and-mouth disease, bluetongue disease, maedi-visna, orf, Tick-borne encephalomyelitis, peste des petits ruminants, sheep pox, and goat pox, as well as bacterial diseases, namely, blackleg, foot rot, caprine pleuropneumonia, contagious bovine pleuropneumonia, Pasteurellosis, mycoplasmosis, streptococcal infections, chlamydiosis, haemophilosis, Johne's disease, listeriosis, and fleece rot [3–10].

The respiratory diseases represent 5.6 per cent of all these diseases in small ruminants. Small ruminants are especially sensitive to respiratory infections, namely, viruses, bacteria, and fungi, mostly as a result of deficient management practices that make these animals more susceptible to infectious agents. The tendency of these animals to huddle and group rearing practices further predispose small ruminants to infectious and contagious diseases [6, 9]. In both sheep and goat flocks, respiratory diseases may be encountered affecting individuals or groups, resulting in poor live weight gain and high rate of mortality. This causes considerable financial losses to shepherds and goat keepers in the form of decreased meat, milk, and wool production along with reduced number of offspring. Adverse weather conditions leading to stress often contribute to onset and progression of such diseases. The condition becomes adverse when bacterial as well as viral infections are combined particularly under adverse weather conditions. Moreover, under stress, immunocompromised, pregnant, lactating, and older animals easily fall prey to respiratory habitats, namely, Streptococcus pneumoniae, Mannheimia haemolytica, Bordetella parapertussis, Mycoplasma species, Arcanobacterium pyogenes, and Pasteurella species [2, 4, 7–9, 12, 13]. Such infections pose a major obstacle to the intensive rearing of sheep and goat and diseases like PPR, bluetongue, and ovine pulmonary adenomatosis (Jaagsiekte) adversely affect international trade [2, 9, 10, 13], ultimately hampering the economy.

Insect-borne diseases are infectious diseases spread among animals and human through hemophagia by blood-feeding arthropods, such as mosquitoes, ticks and midges, which are widespread in the environment. These infections lead to significant human and animal morbidity and mortality worldwide, which cause a huge economic loss. In recent years, epidemic outbreaks of insect-borne infectious diseases in countries neighboring and trading with China have posed a threat to public health, especially in the border areas of China. Many insect-borne diseases in the border areas of China have drawn public attention, such as West Nile virus (WNV) infection in Xinjiang, Tahyna virus infection in the Qinghai-Tibet Plateau, and mosquito-borne arbovirus (e.g. Japanese encephalitis virus and Sindbis virus) infection in Yunnan. It is remains difficult to develop effective vaccines against such viruses. Furthermore, the clinical symptoms of many infections by mosquito-borne arboviruses do not indicate a specific pathogen, and some infections are even asymptomatic. Therefore, accurate and timely diagnosis of these infections is a great challenge of utmost importance.

To date, multiple molecular detection methods have been established to detect insect-borne pathogens, including reverse transcriptase-polymerase chain reaction (PCR), real-time PCR, a liquid bead array and a microwell membrane array. Recent studies have established modified PCR or array methods for the detection of insect-borne pathogens [11–13]. However, these methods are only able to detect one type or a few types of viruses, which greatly restricts their application. Luminex xMAP technology is a multiplexed high-throughput detection system that uses fluorescent carboxylated microspheres. The Luminex array offers up to 100 independent channels and use microspheres embedded with various ratios of two fluorescent dyes. A mixed suspension of microspheres is mixed with the sample to bind analytes, which are then labeled with a fluorescent reporter and analyzed using a specialized flow cytometer [14, 15].

In this study, we established a method that was able to simultaneously detect multiple insect-borne pathogens rapidly and effectively, including bluetongue virus (BTV), epizootic hemorrhagic disease virus of deer (EHDV), Q-fever pathogen Coxiella burnetii (CB), African swine fever virus (ASFV), West Nile fever virus (WNV), Borrelia burgdorferi (BB), vesicular stomatitis virus (VSV), Rift Valley fever virus (RVFV), Ebola virus (EBV) and Schmallenberg virus (SBV). This optimized liquid array detection system may contribute to the rapid and effective detection of multiple insect-borne diseases at border ports in China.

Infectious disease can be viewed as a play involving at least two characters: the pathogen and the host. While both roles can be represented by a great variety of performers, pathogens exhibit by far the highest variety and complexity. This review is about viral infections in animals. It aims first to give an idea of the enormous complexity and diversity of the existing infectious agents, emphasizing their extraordinary capacity for change and adaptation, which eventually leads to the emergence of new infectious diseases. Secondly, it focuses on the influence of the environment in this process, and on how environmental (including climate) changes occurring in recent times, have precise effects on the emergence and evolution of infectious diseases, some of which will be illustrated with specific examples. Finally, it describes the recent and dramatic expansion of two of the most important emerging animal viral diseases at present, bluetongue (BT) and West Nile fever/encephalitis (WNF), dealing with their relationship to climate and other environmental changes, particularly those linked to human activities, collectively known as “global change,” and that can be at least in part seen a consequence of the “globalization” phenomenon.

Almost 120 years have passed since Walter Reed, James Carroll, Aristides Agramonte, and Jesse Lazear established that yellow fever is caused by a filterable infectious agent which is transmitted by the bite of a mosquito, then known as Stegomyia fasciata (Aedes aegypti). Lazear, who like his colleagues, had been stationed by the US Army in Cuba to study the disease, died of yellow fever in September 1900 after being exposed experimentally to mosquitos that had fed on sick patients. At about the same time in South Africa, James Spreull and Sir Arnold Theiler demonstrated that bluetongue disease of sheep is caused by an “ultravisible” agent that could be transmitted by the injection of an infected serum. Epidemiological evidence suggested that the agent was vector-borne, and it was subsequently shown by R.M. du Toit that the disease occurred in sheep inoculated experimentally with suspensions of wild-caught biting midges (Culicoides imicola). These and other seminal discoveries precipitated a century of research into vector-borne and zoonotic viral diseases, resulting in the discovery and isolation of many hundreds of novel viruses from insects or vertebrate hosts. Some were identified as important human or veterinary pathogens. Many other viruses were archived in reference collections, with only basic characterization of their biological or molecular properties. In recent years, the advent of next generation sequencing (NGS) has transformed this situation. Complete genome sequences are now available for many of the archived isolates, allowing more accurate taxonomic assignments, analysis of their phylogenetic and evolutionary relationships with other viruses, and evaluation of the potential risks they may present to humans and wild or domestic animal populations. NGS has also opened the door to viral metagenomics, which has greatly increased the pace of new virus discovery from a wide range of hosts, usually with complete or near-complete viral coding sequences, but no virus isolate and minimal biological data. This has presented both opportunities and challenges for virologists and epidemiologists, as well as viral taxonomists, evolutionary biologists, and bioinfomaticians. Sadly, this technological revolution has been accompanied by a period of progressive disinvestment in training in classical virology. In this review, we recall the rich history of the discovery of arboviruses and other zoonotic viruses in various settings around the world and the many outstanding scientists who have contributed to the endeavour. We also consider the impacts of NGS and metagenomic analysis, and the implications of these new technologies for the future of this important field of research.

Amongst pathogens, RNA viruses were a major source of emerging diseases during the last 30 years. High mutation rate and in case of segmented genome, reassortment are responsible for genetic adaptability and variability of these viruses.

Two pathogens affecting cattle and sheep were responsible for major outbreaks in Mainland Europe in the past 15 years: Bluetongue virus (BTV) and Schmallenberg virus (SBV). These outbreaks were singular in several ways: the diseases were previously either never reported in such northern locations (bluetongue virus) or recently discovered (Schmallenberg virus); their emergence still has unexplained aspects; both viruses displayed the ability to cross the placental barrier. Moreover, these events confirmed that palearctic endemic Culicoides species contribute to the spread of BTV and SBV and to the epizootic aspect of the diseases.

Bluetongue virus causes the eponymous bluetongue disease (BT). BTV belongs to the family Reoviridae, subfamily Sedoreovirinae, and represents the type specie of the Orbivirus genus. The family Reoviridae currently contains fifteen genera of multi-segmented dsRNA viruses, including pathogens of a wide range of vertebrates (including humans), arthropods, plants, and fungi. Unlike the other reoviruses, all orbiviruses are arthropod-borne viruses (arboviruses). This genus currently contains 22 species as well as 10 unclassified “orbiviruses”.

Until recent nomenclature changes implemented by the International Committee on Taxonomy of Viruses Schmallenberg virus was part of the Bunyaviridae family, genus Orthobunyavirus, grouped within the serogroup Simbu along with at least 27 other virus species. The members of the Simbu serogroup show cross-reactions to the complement fixation test but are distinguished by seroneutralization and by genetic sequence analysis. Yet still part of the Orthobunyavirus genus, SBV, AKAV and Aino virus (AINOV) are now considered exemplar viruses of the species Sathuperi orthobunyavirus, Akabane orthobunyavirus, and Shuni orthobunyavirus, respectively. These belong to the new order Bunyavirales, family Peribunyaviridae (formerly Bunyaviridae), which comprises the genus Orthobunyavirus and Herbevirus (host range limited to insects).

Despite their belonging to different viral families, BTV and SBV have several features in common. These converging aspects warrant the present work discussing more specifically the elements to consider while designing experimental infections targeting ruminant host species. A particular emphasis will be given to placental crossing and teratogenic potential of these two viruses.

Since the coincidental isolation of Seneca Valley virus (SVV), recently termed Senecavirus A (SV-A), as a cell culture media contaminant in 2002, a number of serologically similar viruses were identified and grouped to the classification of Senecavirus. The primary sequence analysis of the conserved polypeptide regions (P1, 2C, 3C and 3D) of the first isolate (SVV-001) showed that the virus is most closely related to cardioviruses in the family of Picornaviridae. The single-stranded RNA genome of SV-A displays the secondary structural features of an internal ribosome entry site (IRES) that resembles the IRES element of classical swine fever virus (CSFV) of the family Flaviviridae, giving rise to the possibility that genetic exchange may have occurred between members of Picornaviridae and Flaviviridae during persistent co-infection in pigs. Importantly, SV-A is a natural oncolytic agent, with the ability to selectively replicate in; and kill human tumor cells of neuroendocrine origin, thus, the virus is being advanced as a tool for potential therapeutic intervention of cancer.

Swine are considered to be the natural hosts of SV-A and all known SV-A sequenced isolates have been obtained from pigs. Previously, by regression analysis of partial genome sequences, it was suggested that different isolates of SV-A had a common ancestor and were assumed to have been introduced into the US pig populations (http://www.europic.org.uk/Europic2006/posters/Knowles.svv.01.pdf). Virus isolated in cell culture from tissue specimens of a diseased pig presenting vesicular lesions on the snout and feet in 2005, was identified by the National Veterinary Services Laboratories’ (NVSL) Foreign Animal Disease Diagnostic Laboratory (FADDL) as SV-A using a broad pan-viral microarray (unpublished data). More recently, this vesicular disease syndrome, with as yet unidentified etiology, has been termed swine idiopathic vesicular disease (SIVD) [5, 6]. Despite the isolation of SV-A in cell culture, FADDL has been unsuccessful at reproducing clinical signs by experimental inoculation of pigs with live virus. Negative observations were also made by other laboratories who conducted animal inoculations with multiple SV-A isolates. Singh et al (2012) proposed SV-A as the causative agent of SIVD from a detailed clinical, diagnostic and histopathological study on a Chester White boar suffering from anorexia, lethargy, lameness and vesicular lesions. However, association of SV-A with SIVD, or as the sole causative agent, is speculative at this time since the virus has also been isolated from pigs lacking clinical disease. SIVD has been reported in pigs in the continents of North America and Australia [6, 9–11]. Although SIVD itself does not pose an economic concern, veterinary diagnosis from clinical signs is complicated since similar vesicular lesions can be formed due to common viral infections such as parvovirus, enterovirus, toxins in food supply, or burns [12–16]. Additionally, SIVD clinically resembles high consequence transboundary animal diseases (TADs) such as foot and mouth disease (FMD), swine vesicular disease (SVD), vesicular stomatitis (VS), and vesicular exanthema of swine (VES). A few laboratory methods have been developed for detection of SV-A including a virus serum antibody neutralizing test and a competitive enzyme linked immunosorbant assay (cELISA), which are not widely available [7, 17]. The principal aim of this study was to develop a specific real-time RT-PCR (RT-qPCR) assay for fast, sensitive, and quantitative detection of SV-A RNA in vesicular diagnostic tissues.

Since the beginning of modern virology in the 1950s, transmission electron microscopy (TEM) has been one of the most important and widely used techniques for the identification and characterization of new viruses. Two TEM techniques are usually used for this purpose: negative staining on an electron microscopic grid coated with a support film and (ultra) thin section TEM of infected cells, fixed, pelleted, dehydrated, and embedded in epoxy plastic. Negative staining can be conducted on highly concentrated suspensions of purified virus or cell culture supernatants. For some viruses, TEM can be conducted on contents of skin lesions (e.g., poxviruses and herpesviruses) or concentrated stool material (rotaviruses and noroviruses). For successful detection of viruses in ultrathin sections of infected cells, at least 70% of cells must be infected, and so either high multiplicity of infection (MOI) or rapid virus multiplication is required.

Viruses can be differentiated by their specific morphology (ultrastructure): shape, size, intracellular location or, for some viruses, from the ultrastructural cytopathology and specific structures forming in the host cell during virus replication. Usually, ultrastructural characteristics are sufficient for the identification of a virus at the level of a family. In certain cases, confirmation can be obtained by immuno-EM performed either on virus suspension before negative staining or on ultrathin sections. This requires virus-specific primary antibodies, which might be not available in the case of a novel virus. For on-section immuno-EM, OsO4 post-fixation must be omitted and the partially dehydrated sample must be embedded in a water-miscible acrylic plastic (usually LR White). The ultrastructure of most common viruses is well documented in good atlases and book chapters and many classical publications of the 1960s, 1970s, and 1980s. Several excellent reviews were recently published on the use of TEM in the detection and identification of viruses.

Bluetongue (BT) is a non-contagious, arthropod-borne viral disease of domestic and wild ruminants, listed as a notifiable disease by the World Organization for Animal Health (OIE). The Bluetongue virus (BTV) is the prototype member of the Orbivirus genus within the Reoviridae family (1, 2).

BTV is a double-stranded (ds) RNA virus (3), its genome consists of ten segments (Seg-1 to Seg-10) of linear dsRNA coding 7 structural (VP1–VP7) and 5 non-structural (NS1, NS2, NS3/NS3a, NS4, and NS5) proteins (4) At present twenty-eighth distinct BTV serotypes have been officially recognized based on Seg-2 gene sequence (5, 6). Other putative novel BTV serotypes have also been described (7–11).

Transmission between mammalian hosts and spread of the infection rely mostly on competent Culicoides species (12–14), so the presence of the disease is then strictly related to the distribution of competent vectors (2). Even though they don't seem to be epidemiologically important, vertical and horizontal transmissions have also been described (15–18). For BTV-26, BTV-27 v02 and, probably, BTV-X ITL2015 and BTV-28 transmission by direct contact has been demonstrated or hypothesized (6, 10, 19, 20).

Clinical signs of BT are more severe and most commonly observed in sheep or in white-tailed deer, often leading to animal fatality especially in naïve animals (1, 2). In cattle, BTV infection is usually asymptomatic, although symptoms were reported after infection with some strains (21–24).

As a RNA virus with a segmented genome, BTV can undergo reassortment which can occur when a cell is simultaneously infected with more than one BTV strain and involves the packaging, into a single virion, of full length of genomic segments of different ancestry. Reassortment in BTV is very flexible, and can involve any genome segment (25–27). However, the genome sequence of BTV isolates generally reflects their geographic origins (28–30).

Seg-2 and Seg-6 encoded the BTV outer capsid proteins VP2 and VP5. They represent the primary target for neutralizing antibodies generated during infection of the mammalian host (31–33). Their highly variable sequences are associated with virus serotype (particularly in VP2/segment) (34–36) and, within each serotype, with the geographic origin of the virus strain (28, 34–37).

Among BTV serotypes, Seg-2/VP2 sequences of BTV-3, BTV-16, and BTV-13 are closely related. All of them are included within the nucleotype B reflecting a serological relationship (3). BTV-3 strains can be subdivided into at least two main clusters. 1st cluster includes strains originated from Africa, Mediterranean Basin and North America (western topotypes, w); 2nd-includes strains originated from Japan, India, and Australia (eastern topotypes, e). The nucleotide (nt) identity between Western and Eastern topotype Seg-2 can be as small as 71.5% (7, 38–43).

Between 2016 and 2018, two novel BTV-3 western strains have been identified in two different geographical areas of Tunisia-one in the north-eastern part of the country (Peninsula of Cap Bon, prototype BTV-3 TUN2016) and the other in the South-East near by the border with Libya (prototype strain BTV-3 TUN2016/Zarzis). The BTV-3 TUN2016 spread in 2017 to Italy infecting a single 3-year-old female crossbred sheep belonging to a flock located in the municipality of Trapani, Sicily, which are 150 km distant from Peninsula of Cap Bon (7, 39, 40) and in 2018 in the Southern area of Sardinia causing numerous outbreaks (38). Clinical signs in infected sheep included depression, fever, nasal discharge, submandibular edema, and crusted discharge around the nostrils. Four animals died because of the severity of infection (38). In 2016, another BTV-3 strain closely related to TUN2016/Zarzis strain was detected in Egypt (38).

The present paper reports the results of the diagnostic activities on BTV conducted at Kimron Veterinary Institute between 2013 and 2018 and the evidence of BTV-3 circulation in Israel. Clinical signs of infected sheep, goats and cattle along with the genetic characterization and phylogenetic analysis of the BTV-3 strains are also described.

West Nile virus (WNV) is a positive sense, single-stranded RNA virus and a member of the genus flaviviruses, Family Flaviviridae. In nature, birds are the sylvatic reservoir for WNV in an endemic cycle with the primary vector, the mosquito. The virus is wide spread in Africa, the Middle east and Russia, and since the 1999 outbreak in New York, WNV has spread from east to west in most states in North America. Transmission of the virus to humans via mosquitoes can cause significant health problems such as West Nile fever and a neuroinvasive disease. Unfortunately, no antivirals or vaccines are currently available, and therefore efficient and safe antivirals are urgently needed.

The 11 Kb genome of WNV has a single, open reading frame, which is translated into one polypeptide (in the order:C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5) and processed into 3 structural and 7 non-structural proteins. The viral NS3 protease mediates post-translational modification of the polyprotein in the cytoplasm and by host proteases in the endoplasmic reticulum. WNV replicates in various types of cells in tissue culture, including Vero E6, BHK, and insect cell lines. During replication, host cells may show a cytopathic effect (CPE) from apoptosis. WNV infection can induce apoptosis in several different cell lines, including, insect cells and mammalian cells, possibly through the bax gene.

Development of assays and high throughput (HT) screening platforms for WNV has followed a path similar to a number of other viruses with a focus on cell-based replicon reporter assays and biochemical assays. WNV replicons have been created which harbor luciferase or GFP alone or replicons with luciferase and Neor reporters. The replicons were adapted to a 96-well format, and the assay was validated with known inhibitors of WNV. The first HT screen of WNV consisted of diverse set of 200 compounds in which triaryl pyrazoline was identified as an inhibitor of viral RNA synthesis. In another study, a small library of 108 compound pyrolizines were identified which may inhibit RNA synthesis. In 2006, Gu et al., published a screen of a library of over 35,000 small molecule compounds with the WNV replicon with the luciferase and Neor reporters. They also developed a counterscreen in a 96-well format with live virus which was employed to screen 23 compounds. From these screens, several candidate compounds were identified with one class, a pyrazolopyrimidine, showing promising activity and selectivity as well as promise for follow-up chemistry.

We have developed a cell based assay using WNV (NY-99 strain) in 384-well format that builds from our prior success with live viral HT screens for the influenza virus and SARS CoV. The assay employed cell viability as the end point, using Promega’s CellTiter-Glo®, which produces a luminescent signal in relation to the quantity of ATP in host cells (directly related to cell viability). In contrast to dye formation or dye-uptake methods, which have a low dynamic range, the CellTiter-Glo® assay shows a higher dynamic range with less background. We implemented the WNV assay in the 384-well format in a screen of 13,001 compounds. A diverse group of small molecules targeting various specific steps in virus replication was discovered in the screening campaign.

The Reoviridae family is a large and diverse group of nonenveloped, icosahedral viruses with genomes composed of 9–12 linear molecules of double-stranded RNA (dsRNA). Reoviruses are divided between the Spinareovirinae subfamily (species with turrets on the core particle) and Sedoreovirinae subfamily (species with smooth, nonturreted core particles). They infect numerous host species, from plants to crustaceans, insects, aquatic and terrestrial vertebrates. Among the 16 Reoviridae genera, the Orbivirus genus (subfamily: Sedoreovirinae) is the largest, having 22 species recognized by the International Committee on Taxonomy of Viruses (ICTV) and a significant number of species proposals. Orbiviruses are vector-borne pathogens, primarily transmitted by ticks and other hematophagous insects (mosquitoes, Culicoides biting midges and sand flies). Their wide host range includes wild and domestic ruminants, camelids, equids, humans, marsupials, bats, sloths and birds. The most studied orbiviruses are the Culicoides-borne Bluetongue virus (BTV, type species), African horse sickness virus (AHSV) and Epizootic hemorrhagic disease virus (EHDV), all known as important pathogens of livestock and wildlife. Some orbiviruses such as Tribeč virus, Kemerovo, Lebombo and Orungo viruses have been detected in human infections and are considered human pathogens.

Orbiviral genomes consist of 10 linear segments of dsRNA designated by their decreasing molecular weight. They encode seven structural proteins (VP1–VP7) and three to four nonstructural proteins (NS1, NS2, NS3/NS3a and NS4). The high conservation degree of certain structural core proteins (e.g., polymerase, major core and subcore proteins) recommends them for comparative and phylogenetic analyses of different Orbivirus species. In contrast, the proteins of the outer capsid are highly variable and their specificity to the host’s neutralizing antibody response can be used to distinguish between different serotypes of the same orbivirus species. The phylogenetic clustering of Orbivirus members results in clades indicating their putative or potential arthropod vectors: Culicoides- or sand fly-borne (C/SBOV), mosquito-borne (MBOV) and tick-borne orbiviruses (TBOV). One exception to this classification is St. Croix River virus (SCRV), a distant member of the genus considered to be a “tick orbivirus” (TOV), having no known vector.

As one of Europe’s largest wetlands, the Danube Delta Biosphere Reserve (DDBR) located in the southeast of Romania, is a very biodiverse and heterogeneous complex of ecosystems. The region is a major hub for bird migration along main African–Eurasian fly corridors, with ecoclimatic conditions suitable for abundant and diverse populations of arthropod vectors, which may allow pathogen import and maintenance.

During an arbovirus survey in DDBR, we identified a novel orbivirus in grass snakes (Natrix natrix Linnaeus 1758), tentatively named Letea virus (LEAV) after the eponymous village from the study area. The aims of this study were to characterize the genome of LEAV and its evolutionary relationship with other members of the Orbivirus genus. This is the first report of reptiles as orbivirus hosts. The present study expands our knowledge of orbivirus host range, ecology and the complete genomic data may help understand the evolutionary relationship among species of the Orbivirus genus.

Bluetongue virus (BTV) first emerged in the European Union (EU) in 2006, peaking at 45,000 cases in 2008. The EU spent million of euros in 2008 and 2009 on eradicating and monitoring programs, co-financed with member states. The number of cases in 2009 was 1118, with only 120 reported across the EU so far last year. Vaccination has proven itself as the most effective tool to control and prevent the disease and to facilitate the safe trade of live animals.

Mammalian cells are commonly used as a substrate for production of most of the viral vaccines. BHK-21 cells are used for the production of bluetongue vaccines. Most companies use roller bottles or conventional bioreactors, but taking into account that the manufacturing cost is very important, the possibility of using Single-Use Bioreactor (SUB) technology as an alternative to roller bottles or conventional bioreactors was explored. Advantages like yields of production, time reduction (elimination of cleaning and sterilization steps needed for bioreactors, no validation process, etc) and quality of antigen production were studied.

As can be seen by a graph depicting the number of PubMed citations per year, advances in virology and prion studies are accelerating at a pace that makes it difficult for any individual to remain informed in areas outside one’s specialty (Figure 1). In addition, scientific meetings are continuing to focus on specific areas to maximize dissemination of information to select groups while general meetings that cover multiple fields are typically too large to permit prolonged informal discussion, especially with students and early-stage investigators. With these facts in mind, the Rocky Mountain Virology Association (RMVA) was formed to provide a venue that permitted formal presentations of current research in multiple areas of virology and prion biology in a venue that is sufficiently removed from major cities to ensure extended informal discussions and opportunities to establish/strengthen collaborations. Professional child daycare was provided to help enable attendance by individuals with young children, which provided an educational opportunity through the children’s participation in a virus and prion themed performance during the formal poster session. Taken together, the 17th annual RMVA meeting (Figure 2) upheld the tradition of presenting novel findings, summarized below, that spanned the fields of bio-informatics, host-pathogen interactions, immunology, therapeutics, replication of DNA and RNA viruses along with prion detection and disease propagation.

The basic reproduction ratio (R0) is the most widely used parameter in epidemic theory and is a key tool for understanding the behaviour of infectious diseases. It is defined as the average number of secondary cases produced when a single infected individual is introduced into a fully susceptible population. As the epidemic progresses, as a result of the depletion of susceptible animals or the application of control measures, the basic reproduction ratio changes to the case reproduction ratio (Rt), i.e. the average number of secondary cases arising from a single infected case at time t (for t>0). Knowledge of Rt is very relevant for the control of the epidemic. If Rt<1, each case will, on average, produce less than one secondary case, and the epidemic will tend to die out, even if no further measures are applied. However, if Rt>1, each case will, on average, produce more than one secondary case, and extra measures will be needed to control the epidemic. The proportion of the population that would need to be protected to achieve the eradication of the disease (p) can be estimated as: p>1-(1/Rt). Therefore, the higher the value of Rt, the more difficult it will be to control the epidemic. Both R0 and Rt are usually derived from explicit deterministic susceptible-infectious-recovered (SIR) models, and estimated by fitting a equations to epidemic (case or seroprevalence) data. However, there are two problems associated with this approach. First, many assumptions need to be made, for example about the size of the susceptible population, which in the context of an epidemic is continuously expanding. Second, R0 and Rt are estimated as mean values, without consideration of how these parameters vary over both space and time, thereby excluding spatio-temporal information of considerable epidemiological importance. To overcome these difficulties, Haydon and collaborators developed a method based on the construction and analysis of epidemic trees, which has the advantage that the case-reproduction ratio can be estimated directly from epidemic data.

Bluetongue is a viral disease caused by Bluetongue virus (BTV), which belongs to the genus Orbivirus within the family Reoviridae. Traditionally, 24 different serotypes have been classified, but in recent years, two new serotypes BTV-25 and BTV-26 have been identified, reflecting the dynamic nature of this disease. Although bluetongue affects all ruminant species, severe disease is mainly restricted to certain breeds of sheep. BTV is transmitted between hosts almost exclusively by the bites of certain species of Culicoides biting midges (Diptera: Ceratopogonidae).

In 2007, Andalusia, the southernmost region of Spain, was affected by a devastating epidemic caused by bluetongue virus serotype 1 (BTV-1) resulting in more than four thousand infected farms. Besides BTV-1, Andalusia has been affected by several other BTV serotypes: BTV-10 was introduced in 1956, BTV-4 in 2004 and BTV-8 in 2008. Except in the case of BTV-8, the other serotypes were likely to have been introduced through wind transportation of infected Culicoides from the North of Africa. Introduction of bluetongue from Northern Africa into Southern Spain is considered to be one of the main routes of introduction of new serotypes of bluetongue into Europe. Therefore, understanding the pattern of BTV spread in Andalusia is critical for facilitating effective surveillance and control of future epidemics.

The objective of this study was to calculate the between-herd case-reproduction ratio (Rt) of the BTV-1 epidemic in Andalusia in 2007 using epidemic trees, and to describe the main factors that determined spatial and temporal variation in its value.

During years 2013, 2016, 2017, and 2018, 3,149 samples (714 in 2013, 669 in 2016, 744 in 2017, and 1,022 in 2018) from domestic and wild ruminants were collected and examined at the Kimron Institute, Beit Dagan, Israel (KVI). Samples included whole blood from symptomatic animals, spleen or/and lung samples from dead animals and spleen, lung, placenta and brain samples from aborted fetuses. Details on number, samples, species from which samples were collected are shown in Table 1.

Bluetongue virus (BTV), a double-strand RNA (dsRNA) virus, is the prototype virus in the genus Orbivirus within the family Reoviridae. The segmented BTV dsRNA genome encodes ten viral proteins (VP), including seven structural proteins (VP1-7) and three non-structural (NS) proteins, NS1, NS2 and NS3,,. As a member of arboviruses, BTV is transmitted by certain species of Culicoides biting midges, including C. variipennis and C. imocola

[4],. Bluetongue disease is a non-contagious viral disease affecting domestic animals including sheep and cattle primarily, as well as wild ruminants such as buffalo, antelope, deer, elk and camels. BTV disease is one of the most important diseases of domestic livestock, causing $3 billion per year loss worldwide,. In sheep, the disease is acute, and mortality is accordingly high. BTV is listed under the Office International des Epizooties (OIE) Terrestrial Animal Health Code –2009. Exotic BTV is also listed in the “USDA High Consequence Livestock Pathogens." Due to its economic significance, BTV has been the subject of extensive molecular virology and structural biology studies,,. The recent development of the BTV reverse genetics system facilitates our understanding toward the BTV viral life-cycle, the structural and functional interrelationship among viral proteins,,. Hence, BTV is now one of the well-characterized viruses,,.

The main prevention and control measures in endemic areas include active surveillance programs, animal quarantine and movement restrictions, vaccination and insect control measures,,,. Vaccination is used as the most effective and practical measure to minimize losses related to BTV disease and to potentially interrupt the cycle from infected animal to vector,,. However, due to the complexity of the virus, including the twenty-four different serotypes that have the ability of causing variable diseases, BTV is still endemic in many regions despite the high vaccine coverage in sheep and cattle,,,. The 1998–2001 outbreak of BTV-8 in the Mediterranean Basin is the greatest epizootic of the disease on record, showing that BTV has extended its range northwards into areas of Europe that were never affected before, and has since persisted in many of these locations,,,. In 2006, BTV-8 has reached three northern Europe countries, including the Netherlands, Belgium, and Germany,, and further spread to surrounding countries, reaching Switzerland by the end of October 2007,,,.

Vaccination of individuals/animals during an outbreak can prove effective, however, protection of an individual/animal from the threat may not occur for two or more weeks after the initial vaccination. Hence, only a drug can be offered as a therapeutic treatment of individuals/animals in an endemic area. Surprisingly, there are no antiviral currently available against BTV disease. Recently, anti-BTV drug discovery has been implemented and potential antiviral lead compound(s) has been identified. The development and validation of a cytopathic effect (CPE)-based assay led to the screening of the NIH Molecular Libraries Small Molecule Repository (MLSMR), with 194,950 small molecule compounds then. Further studies, using various primary, secondary and confirmatory assays, confirmed 185 structures that were grouped into six analog series corresponding to six scaffolds enriched within the active set compared to their distribution in the library. Based on the results from the previous studies, we selected and evaluated the virostatic efficacy of a cluster of active compounds, in particular, the aminothiophenecarboxylic acid derivatives. Furthermore, aiming to understand their mechanism of action, various studies were carried out to determine which viral life stage(s) these compounds acted on, focusing on how these virostatic compounds protect cells from BTV-induced apoptosis.

With all the probabilities calculated already, we can calculate the total probability of entry of the disease j from the country i to the European Union, taking into account all the routes of entry already evaluated(PIij).

To do this, we calculate the probability of occurrence of the opposite case, the probability of no introduction of the j disease by any of the routes of entry, using the following formula:

With the same type of formula, it is estimated the likelihood of entry of a disease j in the European Union.

A high, moderate and low risk of introduction of infectious diseases from different countries has been estimated based on a 75 and 90-percentile (P75 and P90) over the final results of probability of each route of entry. Therefore, the results that are over the 75-percentile and 90-percentile are classified as moderate and high risk of entry.

The probability of introduction of the j disease into the European Union through live animal's trade from the country i

(PIAji) is calculated as the proportion of animals that are annually transported to Europe coming from the country i multiplied by the probability of the country i being affected by the disease j

(PPAij).

Bluetongue (BT) is a major non-contagious disease of ruminants transmitted by biting midges of the Culicoides genus. Bluetongue virus (BTV), the etiological agent of BT, is the type species of the Orbivirus genus, in the family Reoviridae [1–3]. Historically, the epidemic distribution was limited to tropical and warm temperate regions where the populations of Culicoides and the BTV replication cycle were both favored by the warm climate. Since 2006, BTV has spread extensively into several unexpected areas including Southern and Northern Europe, resulting in a serious economic burden [4–7].

A complex non-enveloped virus, BTV has a genome consisting of 10 segments of double-stranded RNA (dsRNA) encoding five different non-structural proteins, NS1, NS2, NS3, NS3A and NS4, as well as seven structural proteins (VP1–7) [8–11]. A BTV particle consists of three successive protein layers which form two capsids. The exterior capsid contains two major structural proteins, VP5 and VP2, while the interior capsid contains another two proteins, VP3 and VP7, and encloses a viral transcription complex composed of VP1 (polymerase), VP4 (capping enzyme), and VP6 (helicase) proteins, as well as the viral genome [8, 10, 12, 13]. The non-structural proteins are mainly involved in virus assembly, replication, trafficking, release and morphogenesis [9–11, 14].

The transcriptome is a whole set of gene transcripts of specific cells, tissues, organs, or complete organisms, which associates the genetic information of the genome and the biological function of the proteome. The interaction between hosts or mammalian cells and pathogens such as Marek’s disease virus, influenza virus, avian leukosis virus subgroups, bovine viral diarrhea virus, avian infectious bronchitis virus, Schmallenberg virus and tick-borne flaviviruses has been studied previously by transcriptome analysis [15–21]. Recently, deep sequencing has been considered to be a potent approach to transcriptome analyses which is superior to conventional methods in terms of repeatability and the false-positive rate, as well as the dynamic scale [22, 23]. In this study, we used Aedes albopictus cells to reveal the transcriptome changes after infection with BTV, given the lack of the Culicoides genome sequence. Following this, several mRNA transcripts were selected to confirm the sequencing data by quantitative reverse transcription–polymerase chain reaction (qRT-PCR). The global transcriptome profiling will provide a deep understanding of BTV pathogenesis and virus–vector interactions.