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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.
Historical information as well as microbial sequencing and phylogenetic constructions make it clear that infectious diseases have been emerging and reemerging over millennia, and that such emergences are driven by numerous factors (Table 1). Notably, 60 to 80 percent of new human infections likely originated in animals, disproportionately rodents and bats, as shown by the examples of hantavirus pulmonary syndrome, Lassa fever, and Nipah virus encephalitis–. Most other emerging/reemerging diseases result from human-adapted infectious agents that genetically acquire heightened transmission and/or pathogenic characteristics. Examples of such diseases include multidrug-resistant and extensively drug-resistant (MDR and XDR) tuberculosis, toxin-producing Staphylococcus aureus causing toxic shock syndrome, and pandemic influenza–.
Although precise figures are lacking, emerging infectious diseases comprise a substantial fraction of all consequential human infections. They have caused the deadliest pandemics in recorded human history, including the Black Death pandemic (bubonic/pneumonic plague; 25–40 million deaths) in the fourteenth century, the 1918 influenza pandemic (50 million deaths), and the HIV/AIDS pandemic (35 million deaths so far),.
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.
ZIKV, an emerging flavivirus, shares common clinical symptoms with DENV and chikungunya virus (CHIKV). The outbreaks caused by these viruses present a large number of diagnostic challenges. The clinical manifestations of ZIKV involve similar clinical symptoms to DENV and CHIKV, which include fever, exanthema, conjunctivitis, retro-orbital headache, and arthralgia (Cardoso et al., 2015). The diagnosis of viral infection has specific management implications for medical personnel. The identification of DENV requires a routine follow-up to examine thrombocytes along with hematocrit, whereas for CHIKV, chronic arthralgia should be assessed due to its high prevalence. In the case of ZIKV, a detailed diagnosis of sexual and maternal-fetal transmission should be performed to confirm the risk of congenital microcephaly in newborn babies (Fauci and Morens, 2016). A variety of arboviral infections (arthropod-borne; DENV is the most common arboviral infection) may have similar clinical presentations; therefore, their circulation may be under-reported if specific diagnostic tools have not been implemented. However, there are several drawbacks in ZIKV diagnosis due to the lack of availability of diagnostic tools and the frequent cross-reactivity of antibodies between flaviviruses, which have resulted in several limitations in the use of serology (Musso et al., 2015). Commonly, no routine testing of virus cultures is performed, and an antigenic detection test is lacking at present (Musso et al., 2015; Saiz et al., 2016).
The symptoms of ZIKV infection usually tend to be mild, and the initial symptoms can escape notice, reducing the opportunity to collect a sample. Although the viremic period has not been completely defined, viral RNA has been detected in serum after the onset of symptoms up to day 10. In addition, RNA particles of ZIKV have been detected in urine over an extended period in the acute phase, leading to the possibility of considering an alternative sample type. Evidence suggests that serum samples should be taken during the first 5 days after the onset of symptoms supported in some more detailed studies (Musso et al., 2015). Symptoms of microcephaly associated with ZIKV during the development of newborns in the uterus have been reported (Oduyebo et al., 2016). For the diagnosis of infant microcephaly, a complete analysis of head circumference is requested (Kallen, 2014), as the diagnostic parameters for severe microcephaly include a head circumference more than 3 standard deviations below the mean (Von der Hagen et al., 2014). Testing should be performed in pregnant women with positive or inconclusive results from ZIKV testing. If diagnostic parameters confirm possibility of congenital ZIKV infection in an infant, further clinical evaluation should be performed in follow-up. Fever is a common presenting symptom in patients testing positive for arboviruses due to their association with multiple illnesses; hence, it is suggested to eliminate differential diagnoses (Kelser, 2016). Patients with DENV and ZIKV present with temperatures >40°C and <38.5°C, respectively. ZIKV is usually self-limiting, with symptoms lasting 2 to 7 days. Jaundice is a distinguishing clinical presentation of yellow fever virus and can aid in identifying patients with ZIKV virus. The presence of nausea, vomiting, and bleeding may be helpful in identifying DENV. Any of the above symptoms in an individual who has been exposed to ZIKV indicates the possibility of ZIKV infection, and immediate serum testing should therefore be performed (Centers for Disease Control and Prevention [CDC], 2015a,b).
Alkhurma hemorrhagic fever virus (AHFV) in humans was discovered in 1994. The first case reported in a butcher from the city of Alkhurma, a district south of Jeddah in Saudi Arabia, died of hemorrhagic fever after slaughtering a sheep. The viral infection has a reported fatality rate of up to 25%. Interestingly, one of the previous reports regarding this disease showed a misunderstanding of the real name of this infection, called Alkhurma, not Alkhumra. Because subsequent cases were diagnosed in patients from the small town known as Alkhurma in Jeddah from where the virus got its scientific name; the name was accepted by the International Committee on Taxonomy of Viruses. Thus, based on evidence, the first case was confirmed to be the butcher, following the slaughtered sheep. Therefore, a study was conducted among affected patients to address this disease as a public health issue. Blood samples were collected from household contacts of patients with laboratory-confirmed virus for follow-up testing by enzyme-linked immunosorbent serologic assay (ELISA) for AHFV-specific immunoglobulin (Ig) G. Samples from persons seeking medical care were tested by ELISA for AHFV-specific IgM and IgG using AHFV antigen. Viral-specific sequence was performed by reverse transcription PCR (TiBMolbiol, LightMix kit; Roche Applied Science, Basel, Switzerland). A total of 11 cases were identified through persons seeking medical care, whose illnesses met the case definition for AHFV, and another 17 cases were identified through follow-up testing of household contacts.
Subsequently, the virus was isolated from six other butchers of different ages (between 24 and 39 years) from the city of Jeddah, with two deaths. The diagnosis was established from their blood sample tests. The serological tests later confirmed four other patients with the disease. From 2001 to 2003, the study on the virus initial identification in the city of Alkhurma again identified 37 other suspected cases; with laboratory confirmation of the disease in 20 (~55%) of them. Among the 20, 11 (55%) had hemorrhagic manifestations and 5 (25%) died. The virus was later identified in three other locations: from the Western Province of Saudi Arabia (Ornithodoros savignyi and Hyalomma dromedarii were found by reverse transcription in ticks) and from samples collected from camels in Najran. AHFV virus was considered as one of the zoonotic diseases; however, the mode of transmission is not yet clear. Recently, it was suggested that the disease reservoir hosts may include both camels and sheep. The virus might also be transmitted as a result of skin wounds contaminated with the blood or body fluids of an infected sheep; through the bite of an infected tick, and through drinking of unpasteurized or contaminated milk from camels.
In humans, this zoonotic disease may present with clinical features ranging from subclinical or asymptomatic features to severe complications. It is related to Kyasanur Forest disease virus, which is localized in Karnataka, India. However, epidemiologic findings suggest another wider geographic location for the disease in western (including Jeddah and Makkah) and southern (Najran) parts of Saudi Arabia, and the virus infections mostly occur in humans. A study was conducted by Alzahrani et al. in the southern part of Saudi Arabia particularly in the city of Najran (with populations of ~250,000), an agricultural city in Saudi Arabia, where domestic animals are reared at the backyard of owners. After the initial virus identification, from January 2006 through April 2009, 28 persons with positive serologic test results were identified. Infections were suspected if a patient had an acute febrile illness for at least two days; when all other causes of fever have been ruled out. Additionally, data analysis indicated that patients infected with the virus were either in contact with their domestic animals, involved in slaughtering of the animals, handling of meat products, drinking of unpasteurized milk, and/or were bitten by ticks or mosquitoes. Symptoms consistent with AHFV infection—including fever, bleeding, rash, urine, color change of the feces, gum bleeding, or neurologic signs—then develop. Fortunately, infected patients responded to supportive care (including intravenous fluid administration and antimicrobial drugs when indicated), with no fatal cases.
In summary, AHFV is a zoonotic disease with clinical features ranging from subclinical or asymptomatic features to severe complications. Another study highlighted different characteristics of the exposure to the blood or tissue of infected animals in the transmission of AHFV to humans. Of the 233 patients confirmed with infections, 42% were butchers, shepherds, and abattoir workers, or were involved in the livestock industry. More recently, a study on infection using C57BL/6J mice cells showed that the clinical symptoms of the disease were similar to the presentations in humans. However, Alkhurma disease resulted in meningoencephalitis and death in Wistar rats, when high titers to the infection occurred. In addition, exposures to mosquito bites are regarded as potential sources of transmissions of the infection; however, very few available data support this. Although, available data shows that Alkhurma virus has been isolated following mosquito bites. However, another study suggested that mosquitoes may play a role only as a vector in the transmission of the disease.
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.
Occult hepatitis B virus infection (OBI) is an emerging type of HBV infection when HBV DNA is detectable among HBsAg negative infected patients (100). Attention to OBI has increased due to its potential role in accelerating the progression of liver fibrosis and cirrhosis, ultimately leading to hepatocellular carcinoma (HCC); It is transmitted via blood transfusion and transplantation (100).
Introduction of the occult infection was the consequence of improvement of HBV DNA detection and introduction of more sensitive methods which were not available earlier than 1985 (101). OBI seems to be highly prevalent in Asia (102). The first evidence of OBI in Iran refers to 2001–2002 when 22% of chronic liver patients were revealed to be positive for HBV genome tests (103). It has been detected amongst 30% of high risk groups as well as hemodialysis patients and is considered common in HIV patients (104–106).
As OBI has been found among a large proportion of HBcAb positive healthy blood donors, thus the blood of these people should be screened in blood transfusion centers to prevent HBV transmission (84, 107–109).
Powassan virus (POWV) is the only North American member of the tick-borne encephalitis complex (TBE-C) of viruses, which are transmitted by the bite of an infected tick. Other members of the TBE-C include the following flaviviruses: tick-borne encephalitis virus (TBEV) in Eastern Europe and Western Asia, Omsk hemorrhagic fever virus in Siberia, Kyasanur Forest disease virus in India, Alkhurma virus in Saudi Arabia, and Louping ill virus in the United Kingdom. TBE-C viruses can cause a wide range of disease in humans, from mild febrile illness with biphasic fever to encephalitis or hemorrhagic fever (1). POWV is composed of two genetically and ecologically distinct lineages (2). Prototype POWV (lineage I) is transmitted primarily by Ixodes cookei, a tick with a narrow vertebrate host range that rarely feeds on humans. Powassan virus lineage II, also known as deer tick virus (DTV), is transmitted by Ixodes scapularis, a tick with a broad host range that also transmits the infectious agents that cause Lyme disease, anaplasmosis, ehrlichiosis, and babesiosis (3). Since the late 1990s, POWV infections have been reported in the Northeastern and Midwestern parts of the United States as well as in Canada, and incidence is increasing (4). Because the territory of I. scapularis is expanding and the prevalence of POWV in ticks and mammals is increasing, POWV poses an increasing threat (5, 6). In a recent survey, I. scapularis ticks collected from the northwest quadrant of Wisconsin from 2011 to 2012 demonstrated a POWV infection frequency of 4.67% (7). This is similar in frequency to a survey conducted in that same area in 1998 (8). Although POWV is rarely diagnosed as a cause of encephalitis, infections can be severe, and neurologic sequelae are common (9). Additionally, the potential for concurrent transmission with other tick-borne pathogens, particularly Borrelia burgdorferi, the causative agent of Lyme disease, has not been previously studied in North America.
Similarly to other arboviral infections, POWV diagnosis is complex, requiring review of clinical and travel history in addition to knowledge of and access to diagnostic testing (10). Serologic testing remains the primary method for diagnosis of POWV infection, with an emphasis on the detection of POWV-specific IgM antibodies in serum or plasma. Until recently, commercial laboratory testing has been unavailable for POWV in the United States. Prior to this, a positive POWV IgM enzyme immunoassay (EIA) result confirmed by plaque reduction neutralization test (PRNT), a 4-fold or greater increase in titers between acute- and convalescent-phase sera, or culture or direct identification of virus-specific nucleic acids at a state public health laboratory or the Centers for Disease Control and Prevention (CDC) (11) has been the mainstay of diagnostic testing.
We describe here a laboratory-developed, serologic test panel, commercially available at a reference laboratory, for the detection of IgG and IgM antibodies to POWV in serum and plasma samples. The first test in the panel is a highly sensitive, commercial TBE-C screen by EIA. Per the manufacturer, cross-reactivity with other flaviviruses is expected, particularly with West Nile virus (WNV) and dengue virus (DENV) antibody-positive samples. Samples that are screen positive are then confirmed for POWV by indirect immunofluorescence assay (IFA). Performance characteristics of the test panel were optimized, and validation studies were performed to assess the analytical sensitivity, reproducibility, and specificity/cross-reactivity of the serologic test panel for use in routine diagnostic testing.
Influenza A virus (IAV) has caused significant morbidity and mortality globally in humans, with an estimated 14 pandemics that have occurred since the 1500s.1 Wild aquatic birds are well known to be the natural reservoirs for IAV subtypes harbouring H1–H16 subtypes,2, 3, 4 with the exception of H17 and H18 subtypes that were recently discovered in bats.5, 6 The phylogenetic relationships of all IAV subtypes are displayed in Fig. 1. In addition to its natural reservoir species, influenza viruses infect a wide range of hosts including canids, equids, humans and swine.2 IAVs’ ability to generate novel gene constellations through reassortment between subtypes poses a risk for immune escape in these new hosts.7 Furthermore, IAV undergoes rapid genetic and antigenic evolution, which makes vaccination control difficult in humans and other domestic species.
In addition to human pandemics that have emerged from avian and swine hosts, there are also repeated spillover events from domesticated animals, primarily poultry and swine, that pose a significant threat to human health.8, 9, 10, 11, 12, 13, 14 Direct transmission of IAV from a wild avian source to humans is rare, as there has only been a single report of laboratory‐confirmed human infection with H5N1 contracted through close contact with dead and infected wild swan in Azerbaijan.15 However, there is serological evidence of H5N1 infection among Alaskan hunters who handled dead wild avian species,16 indicating that exposure to IAVs from wild birds through close contact can potentially cause infection. More notably, viral genes that are similar to the 1918‐like H1N1 avian virus were recently detected in the influenza gene pools of wild birds, raising the potential for the re‐emergence of a 1918‐like pandemic virus.17 Furthermore, due to increasing human encroachment of wildlife habitats, the potential of a wild‐source threat becomes more relevant, as is seen with the emergence of other pathogens such as human immunodeficiency virus (HIV), severe acute respiratory syndrome coronavirus and the more recent Zaire‐variant Ebola virus in Western Africa.18, 19, 20, 21
In this review, we discuss the current knowledge of ecological and molecular determinants responsible for interspecies transmission of IAV, with specific focus on avian‐derived influenza subtypes involved in zoonotic and epizootic transmission to other hosts (see Fig. 2).
Bats and the viruses they harbor have been of interest to the scientific community due to the unique association with some high consequence human pathogens in the absence of overt pathology. Virologic and serologic reports in the literature demonstrate the exposure of bats worldwide to arboviruses (arthropod-borne viruses) of medical and veterinary importance. However, the epidemiological significance of these observations is unclear as to whether or not bats are contributing to the circulation of arboviruses.
Historically, a zoonotic virus reservoir has been considered a vertebrate species which develops a persistent infection in the absence of pathology or loss of function, while maintaining the ability to shed the virus (e.g., urine, feces, saliva). Haydon et al. extended this definition of a reservoir to include epidemiologically-connected populations or environments in which the pathogen can be permanently maintained and from which infection is transmitted to the defined target population. The significance of the relative pathogenicity of the infectious agent to the purported reservoir host has been debated. In the case of bats as a reservoir species, rigorous field and experimental evidence now exist to solidify the role of the Egyptian rousette bat (Rousettus aegyptiacus) as the reservoir for Marburg virus. Considering arboviruses, additional criteria must be met in order to consider a particular vertebrate species a reservoir. Reviewed by Kuno et al., these criteria include the periodic isolation of the infectious agent from the vertebrate species in the absence of seasonal vector activity, and the coincidence of transmission with vector activity. Further, the vertebrate reservoir must also develop viremia sufficient to allow the hematophagous arthropod to acquire an infectious bloodmeal in order for vector-borne transmission to occur. Bats have long been suspected as reservoirs for arboviruses, but experimental data that would support a role of bats as reservoir hosts for certain arboviruses remain difficult to collect. Here we synthesize what information is currently known regarding the exposure history and permissiveness of bats to arbovirus infections, and identify knowledge gaps regarding their designation as arbovirus reservoirs.
Two major categories of emerging infections—newly emerging and reemerging infectious diseases—can be defined, respectively, as diseases that are recognized in the human host for the first time; and diseases that historically have infected humans, but continue to appear in new locations or in drug-resistant forms, or that reappear after apparent control or elimination. Emerging/reemerging infections may exhibit successive stages of emergence. These stages include adaptation to a new host, an epidemic/pathogenic stage, an endemic stage, and a fully adapted stage in which the organism may become nonpathogenic and potentially even beneficial to the new host (e.g., the human gut microbiome) or stably integrated into the host genome (e.g., as endogenous retroviruses). Although these successive stages characterize the evolution of certain microbial agents more than others, they nevertheless can provide a useful framework for understanding many of the dynamic relationships between microorganisms, human hosts, and the environment.
It is also worth noting that the dynamic and complicated nature of many emerging infections often leaves distinctions between emerging and reemerging infections open to question, leading various experts to classify them differently. For example, we describe as “reemerging” new or more severe diseases associated with acquisition of new genes by an existing microbe, e.g., antibiotic resistance genes, even when mutations cause entirely new diseases with unique clinical epidemiologic features, e.g., Brazilian purpuric fever. Similarly, we refer to SARS as an emerging disease a decade after it disappeared, and apply the same term to the related MERS (Middle East Respiratory Syndrome) β coronavirus which appeared in Saudi Arabia in late 2012.
Zika infection is asymptomatic in 80% of cases. When symptomatic, it is typically described as a mild dengue-like illness, manifested by two or more of the following: sudden onset fever, conjunctivitis, arthralgias, and maculopapular rash (Figure 2). Myalgias and headache are also common. The WHO interim case definition describes suspected, probable and confirmed cases.12 Unlike in dengue, death from acute Zika virus infection is rare, but has been reported in a child with sickle cell disease in Colombia.13 The incubation period for Zika virus after a person is bitten by an infected mosquito ranges from 2–7 days, and may be up to 14 days. This is the reason for screening for risk factors within 14 days of symptom onset. The incidence of GBS or microcephaly appears to be similar between symptomatic and asymptomatic cases.
The Zika virus is a mosquito-borne flavivirus with an approximately 11 kilobase ribonucleic acid (RNA) genome.1 The virus usually causes a mild infection in adults, symptoms include fever, arthralgia and rash.2,3 However, severe complications can occur, such as Guillain-Barré syndrome,4 meningoencephalitis,5 hearing loss and uveitis.6,7 In the current Zika virus outbreak, intrauterine infections have been associated with fetal malformations.8–11
Reliable detection of the Zika virus in infected people is key to understanding the epidemiology, the pathogenesis and alternative transmission routes of the virus, such as sexual intercourse and blood transfusions.12 However, in areas where the Zika virus is co-circulating with dengue and chikungunya viruses, physicians cannot reliably diagnose the Zika virus infection by clinical presentation, because the viruses cause similar symptoms. Using serology for Zika virus diagnostics can be challenging because of the cross-reactivity of antibodies elicited by other endemic flaviviruses – such as dengue, yellow fever, St Louis encephalitis and West Nile viruses.3,13,14 Molecular detection of viral nucleic acid using real-time reverse transcription (RT)-polymerase chain reaction (PCR) assay is a highly reliable diagnostic method during acute infection. Currently, there are six widely used real-time RT–PCR assays for Zika virus detection.7,13,15,16 The Pan American Health Organization (PAHO) has recommended an additional real-time RT–PCR assay.12
High real-time RT–PCR sensitivity is important to avoid false-negative results. Nucleotide mutations in the binding sites of primers and probes can affect the sensitivity.17 So far, the genetic variability of the Zika virus Asian lineage causing the current American outbreak is limited to about 2% nucleotide differences across the viral genome (Fig. 1). However, mutations do not occur evenly across viral genomes. Up to 10 nucleotide mismatches between the oligonucleotide sequences of the published assays and the Asian lineage consensus sequence already exist and in individual primers or probes, there are up to five mismatches (Fig. 2). Note that these are worst-case scenarios based on the genetic variability permitted within the Asian Zika virus lineage, with no single known Zika virus strain accumulating all of these mismatches. However, the increasing number of divergent Zika virus outbreak strains highlights the genetic variability as a potential limiting factor of the sensitivity of Zika virus real-time RT–PCR-based diagnostics.
Here, we compare the sensitivity of published real-time RT–PCR assays and two new assays, which we designed to have less nucleotide mismatches with the current outbreak strains. We also present data on viral load profiles in blood and urine from infected patients, using one of the new assays.
Zika virus (ZIKV), the causative agent of the infectious disease Zika fever, is a positive-sense RNA virus that belongs to the family Flaviviridae, genus Flavivirus, and is similar to Dengue virus (DENV), yellow fever virus, Japanese encephalitis virus, and West Nile virus (Sikka et al., 2016). ZIKV was first isolated from Rhesus macaques in Uganda in 1947. Previously, only sporadic cases of negligible concern associated with human ZIKV infection were reported (Hayes, 2009). Now, ZIKV infections have become epidemic throughout the world (Charrel et al., 2016).
In the north-eastern states of Brazil, the public health authorities recently confirmed autochthonous transmission of ZIKV with the first known reported case of ZIKV infection in mainland South America (Campos et al., 2015; Zanluca et al., 2015), followed by 26 countries, including countries in the European Union and the outermost regions of the Americas, such as Barbados, Bolivia, Brazil, Colombia, Costa Rica, Curacao, Dominican Republic, Ecuador, El Salvador, French Guiana, Guadeloupe, Guatemala, Guyana, Haiti, Honduras, Jamaica, Martinique, Mexico, Nicaragua, Panama, Paraguay, Puerto Rico, Saint Martin, Suriname, the US Virgin Island, and Venezuela (Pan American Health Organization [PAHO], 2016; World Health Organization [WHO], 2016). An increased frequency of ZIKV infection among world travelers has been reported in European countries, including Austria, Denmark, Finland, France, Germany, Ireland, Italy, Portugal, the Netherlands, Spain, Sweden, Switzerland, and the UK (European Centre for Disease Prevention [ECDC], 2016).
The virion of ZIKV consists of an approximately 11 kb positive-sense RNA with a single capsid and two membrane-associated envelope proteins (M and E) (Leyssen et al., 2000; Daep et al., 2014; Charrel et al., 2016). Recent outbreaks of ZIKV infections have become fatal on a daily basis in the Americas, where this obscure viral candidate has been placed at the forefront of global healthcare. The reported occurrences of ZIKV infections are thought to be transmitted mainly by the mosquito species Aedes aegypti and Aedes albopictus. Infections have now dramatically increased in highly populated areas of South, Central, and North America due to the increased frequency of the international travel from Zika-infected areas (Bogoch et al., 2016). Considering the calamity of ZIKV infection, there is an urgent need to develop rapid detection methods for ZIKV along with DENV, which shares common clinical symptoms with ZIKV. The purpose of this review is to provide a complete update of the various analytical methods for virus detection, such as molecular, immunological, sensor-based and other detection assays, along with the advantages and limitations of these strategies. Furthermore, we suggest innovative hypothetical approaches for the development of liposome-based rapid detection assays for ZIKV detection, which will provide new insight to medical professionals for controlling this widespread epidemic virus candidate.
Patients should be assessed for pregnancy as well as for neurological symptoms and signs indicating possible GBS. While the situation is rapidly evolving, as of March 2016, laboratory testing is only available through the Centers for Disease Control and Prevention (CDC) and several state and territory health departments. Generally, testing is not recommended for asymptomatic persons, with the exception of pregnant women 2 to 12 weeks after travel to areas with ongoing Zika virus transmission.17 In symptomatic patients, serum should be obtained for polymerase chain reaction (RT-PCR), IgM, and IgG if within seven days of symptom onset. If more than seven days has elapsed, only IgM and IgG should be assessed; RT-PCR is not indicated.18 Note that serological cross reactivity may occur between Zika and other flaviviruses (e.g., dengue, yellow fever, St. Louis encephalitis, Japanese encephalitis, West Nile). Public health departments may decline to test if the clinical scenario is not suggestive of Zika virus or associated complications. In particular, due to the high cross-reactivity with related flaviviruses, serologic test interpretation is complex and can be difficult, leading to false positive results. Conversely, a negative Zika IgM or RT-PCR test result does not rule out Zika virus infection. Emergency clinicians should consult with public health experts to assist with interpretation of test results.
Central nervous system (CNS) infections including meningitis and encephalitis are important causes of significant mortality and morbidity in the developing nations. Viruses are considered as important etiological agents of CNS infections, causing diseases ranging from febrile illness to myelitis to meningoencephalitis. However, in most cases, the etiology of CNS infection is not known due to lack of diagnostic capacity, standard clinical case definitions, or low levels of surveillance. Specific diagnosis for CNS infection is rarely made and usually categorized empirically as only “viral” or “bacterial”. There have been few reports on the viral etiologies of CNS infections in Indonesia except for Japanese encephalitis virus (JEV), a leading cause of acute encephalitis in children and young adults in the Southeast Asian region [2–5]. Still, JEV is significantly underreported in Indonesia. Furthermore, in endemic provinces like Bali where encephalitis is often suspected to be JEV, there is lack of laboratory capability to accurately determine the disease burden of JEV and other CNS viruses. The objective of this study was therefore to detect and identify the pathogens responsible for viral CNS infections amongst in-patients at a referral hospital in Manado, North Sulawesi, Indonesia.
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.
The central nervous system (CNS), a marvel of intricate cellular and molecular interactions, maintains life and orchestrates homeostasis. Unfortunately, the CNS is not immune to alterations that lead to neurological disease, some resulting from acute, persistent or latent viral infections. Several viruses have the ability to invade the CNS, where they can infect resident cells, including the neurons. Although rare, viral infections of the CNS do occur. However, their incidence in clinical practice is difficult to evaluate precisely. For instance, in cases of viral encephalitis involving the most prevalent viruses known to reach the CNS (mainly herpesviruses, arboviruses and enteroviruses), an actual viral presence can only be detected in 3 to 30 cases out of 100,000 persons. Considering all types of viral infections, between 6000 and 20,000 cases of encephalitis that require hospitalization occur every year in the United States, representing about 6 cases per 100,000 infected persons every year. As the estimated charge for each case lies between $58,000 and $89,600, an evaluation of the total annual health cost is of half a billion dollars. Due to the cost associated with patient care and treatment, CNS viral infections cause considerably more morbidity and disabilities in low-income/resource-poor countries.
Very common worldwide, viral infections of the respiratory tract represent a major problem for human and animal health, imposing a tremendous economic burden. These respiratory infections induce the most common illnesses and are a leading cause of morbidity and mortality in humans worldwide, causing critical problems in public health, especially in children, the elderly and immune-compromised individuals. Viruses represent the most prevalent pathogens present in the respiratory tract. Indeed, it is estimated that about 200 different viruses (including influenza viruses, coronaviruses, rhinoviruses, adenoviruses, metapneumoviruses, such as human metapneumovirus A1, as well as orthopneumoviruses, such as the human respiratory syncytial virus) can infect the human airway. Infants and children, as well as the elderly represent more vulnerable populations, in which viruses cause 95% and 40% of all respiratory diseases, respectively. Among the various respiratory viruses, some are constantly circulating every year in the human populations worldwide, where they can be associated with a plethora of symptoms, from common colds to more severe problems requiring hospitalization. Moreover, in addition to the many “regular” viruses that circulate and infect millions of people every year, new respiratory viral agents emerge from time to time, causing viral epidemics or pandemics associated with more serious symptoms, such as neurologic disorders. These peculiar events usually take place when RNA viruses like influenza A, human coronaviruses, such as MERS-CoV and SARS-CoV, or henipaviruses, present in an animal reservoir, cross the species barrier as an opportunistic strategy to adapt to new environments and/or new hosts. These zoonoses may have disastrous consequences in humans, and the burden is even higher if they have neurological consequences.
Epithelial cells that line the respiratory tract are the first cells that can be infected by respiratory viruses. Most of these infections are self-limited and the virus is cleared by immunity with minimal clinical consequences. On the other hand, in more vulnerable individuals, viruses can also reach the lower respiratory tract where they cause more severe illnesses, such as bronchitis, pneumonia, exacerbations of asthma, chronic obstructive pulmonary disease (COPD) and different types of severe respiratory distress syndromes. Besides all these respiratory issues, accumulating evidence from the clinical/medical world strongly suggest that, being opportunistic pathogens, these viruses are able to escape the immune response and cause more severe respiratory diseases or even spread to extra-respiratory organs, including the central nervous system where they could infect resident cells and potentially induce other types of pathologies.
Like all types of viral agents, respiratory viruses may enter the CNS through the hematogenous or neuronal retrograde route. In the first, the CNS is being invaded by a viral agent which utilizes the bloodstream and in the latter, a given virus infects neurons in the periphery and uses the axonal transport machinery to gain access to the CNS. In the hematogenous route, a virus will either infect endothelial cells of the blood-brain-barrier (BBB) or epithelial cells of the blood-cerebrospinal fluid barrier (BCSFB) in the choroid plexus (CP) located in the ventricles of the brain, or leukocytes that will serve as a vector for dissemination towards the CNS. Viruses such as HIV, HSV, HCMV, enteroviruses such as coxsackievirus B3, flaviviruses, chikungunya virus (CHIKV) and echovirus 30 have all been shown to disseminate towards the CNS through the hematogenous route. Respiratory viruses such as RSV, henipaviruses, influenza A and B and enterovirus D68 are also sometimes found in the blood and, being neuroinvasive, they may therefore use the hematogenous route to reach the CNS. As they invade the human host through the airway, the same respiratory viruses may use the olfactory nerve to get access to the brain through the olfactory bulb. On the other hand, these viruses may also use other peripheral nerves like the trigeminal nerve, which possesses nociceptive neuronal cells present in the nasal cavity, or alternatively, the sensory fibers of the vagus nerve, which stems from the brainstem and innervates different organs of the respiratory tract, including the larynx, the trachea and the lungs.
Although the CNS seems difficult for viruses to penetrate, those pathogens that are able to do so may disseminate and replicate very actively and will possibly induce an overreacting innate immune response, which may be devastating. This situation may lead to severe meningitis and encephalitis that can be fatal, depending on several viral and host factors (including immunosuppression due to disease or medications) that may influence the severity of the disease.
Recently, a very interesting manuscript produced by Bookstaver and collaborators underlined the difficulties of precisely deciphering the epidemiology and identifying the causal agent of CNS infections. These difficulties are mainly due to the tremendous variation in the symptoms throughout the disease process and to the myriad of viruses that can cause CNS infections. As stated in their report, these authors underlined that the clinical portrait of viral infections is often nonspecific and requires the clinician to consider a range of differential diagnoses. Meningitis (infection/inflammation in meninges and the spinal cord) produces characteristic symptoms: fever, neck stiffness, photophobia and/or phonophobia. Encephalitis (infection/inflammation in the brain and surrounding tissues) may remain undiagnosed since the symptoms may be mild or non-existent. Symptoms may include altered brain function (altered mental status, personality change, abnormal behavior or speech), movement disorders and focal neurologic signs, such as hemiparesis, flaccid paralysis or paresthesia. Seizures can occur during both viral meningitis and encephalitis. Furthermore, viral encephalitis may also be difficult to distinguish from a non-viral encephalopathy or from an encephalopathy associated with a systemic viral infection occurring outside the CNS. Considering all these observations, it is therefore mandatory to insist on the importance of investigating the patient’s history before trying to identify a specific viral cause of a given neurological disorder.
In humans, a long list of viruses may invade the CNS, where they can infect the different resident cells (neuronal as well as glial cells) and possibly induce or contribute to neurological diseases, such as acute encephalitis, which can be from benign to fatal, depending on virus tropism, pathogenicity as well as other viral and patient characteristics. For instance, 30 years ago, the incidence of children encephalitis was as high as 16/100,000 in the second year of life, while progressively reducing to 1/100,000 by the age of 15. More recent data indicate that, in the USA, the herpes simplex virus (HSV) accounts for 50–75% of identified viral encephalitis cases, whereas the varicella zoster virus (VZV), enteroviruses and arboviruses are responsible for the majority of the other cases in the general population. Several other viruses can induce short-term neurological problems. For example, the rabies virus, herpes simplex and other herpes viruses (HHV), arthropod-borne flaviviruses such as the West Nile virus (WNV), Japanese encephalitis virus (JEV), chikungunya virus (CHIKV), Zika virus (ZIKV), alphaviruses such as the Venezuelan, Western and Eastern equine encephalitis viruses and enteroviruses affect millions of individuals worldwide and are sometimes associated with encephalitis, meningitis and other neurological disorders. The presence of viruses in the CNS may also result in long-term neurological diseases and/or sequelae. Human immunodeficiency virus (HIV) induces neurodegeneration, which lead to motor dysfunctions and cognitive impairments. Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease associated with reactivation of latent polyoma JC virus (JCV). Progressive tropical spastic paraparesis/HTLV-1-associated myelopathy (PTSP/HAM) is caused by human T-lymphotropic virus (HTLV-1) in 1–2% of infected individuals. Measles virus (MV), a highly contagious common virus, is associated with febrile illness, fever, cough and congestion, as well as a characteristic rash and Koplik’s spots. In rare circumstances, significant long-term CNS diseases, such as post-infectious encephalomyelitis (PIE) or acute disseminated encephalomyelitis (ADEM), occur in children and adolescents. Other examples of rare but devastating neurological disorders are measles inclusion body encephalitis (MIBE), mostly observed in immune-compromised patients, and subacute sclerosing panencephalitis (SSPE) that appears 6–10 years after infection.
Yet, with the exception of HIV, no specific virus has been constantly associated with specific human neurodegenerative disease. On the other hand, different human herpes viruses have been associated with Alzheimer’s disease (AD), multiple sclerosis (MS) and other types of long-term CNS disorders. As accurately stated by Majde, long-term neurodegenerative disorders may represent a “hit-and-run” type of pathology, since some symptoms are triggered by innate immunity associated with glial cell activation. Different forms of long-term sequelae (cognitive deficits and behavior changes, decreased memory/learning, hearing loss, neuromuscular outcomes/muscular weakness) were also observed following arboviral infections.
Including the few examples listed above, more than one hundred infectious agents (much of them being viruses) have been described as potentially encephalitogenic and an increasing number of positive viral identifications are now made with the help of modern molecular diagnostic methods. However, even after almost two decades into the 21st century and despite tremendous advances in clinical microbiology, the precise cause of CNS viral infections often remains unknown. Indeed, even though very important technical improvements were made in the capacity to detect the etiological agent, identification is still not possible in at least half of the cases. Among all the reported cases of encephalitis and other encephalopathies and even neurodegenerative processes, respiratory viruses could represent an underestimated part of etiological agents.
Hepatitis C virus (HCV) has affected about 175 million people worldwide and is considered as one of the leading cause of liver transplantation (77, 78). In Iran, the virus is introduced as an emerging viral infection amongst high risk populations like injecting drug users as this group has shown a higher prevalence of HCV in recent years (79).
A study conducted in 1994 on healthy blood donors, revealed 0.25% of seroconversion for HCV infection for the first time in Iran (79). HCV infection prevalence has a low rate in general population in Iran compared to the adjacent countries of Pakistan, Turkey and Iraq (80–82). The infection amongst blood donors is 0.1 to 0.5% in different cities of the country (83–86).
Different dialysis centers have had diverse frequency of HCV infection ranging from 5 to 23.9% (87, 88). The main route of HCV transmission among hemophilia and thalassemia patients is through blood products (89–93). In years 1999 and 2000, 0.59% of HCV antibody positive cases were confirmed in multi-transfused children with β-thalassemia in Shiraz blood bank (94). In 2005, a multicenter study pointed out that 19.3% of thalassemia patients suffered from HCV infection (91). In 2007, the infection rate varied between 15.7% and 63.8% (95). In recent years, other serological studies have shown that 15 to 91% of all patients with hemophilia have antibodies against HCV (96–99). This evidence emphasizes the importance of screening of hemophilic patients for HCV infection.
The HCV genome pattern has changed during recent years in Iran and it seems that such a change can be due to cross-border travels between Iran, Pakistan and Iraq (82).
To decrease the trend of infection, regular surveys and interventions should be done, focusing on high-risk groups such as IDUs, those who receive blood products and health care workers with occupational exposure (99).
This study was approved by the Medical Research Ethics Committee of R.D. Kandou General Hospital (Ethical Approval No. 066/EC-UPKT/III/2016) and Eijkman Institute Research Ethics Commission (Ethical Approval No. 78). Written informed consent for participating in the study was obtained from all of the patients, guardians, or accompanying close relatives.
Countries in the Eastern Mediterranean Region (EMR) continue to be hotspots for emerging and reemerging infectious diseases (1). Outbreaks of such diseases have a significant impact on health and economic development in the Region. At least 11 of the 22 countries in the Region have reported epidemics from emerging infectious disease over the past 10 years with the potential for global spread (2). These epidemic threats remain potentially devastating to the social and economic development of the Region, combined with the risk for international spread. The need to prevent, detect, and respond to any infectious disease that poses a persistent threat to global health security remains a national, regional, and international priority (3).
The mission of WHO Health Emergencies (WHE) Program is to build the capacity of Member States to manage health emergency risks and, when national capacities are overwhelmed, to lead and coordinate the international health response to contain outbreaks and to provide effective relief and recovery to affected populations.
The Infectious Hazard Management unit of WHE program in the EMR of World Health Organization (WHO) is responsible for establishing risk mitigation strategies and capacities for priority high-threat infectious hazards. This includes developing and supporting prevention and control strategies, tools and capacities for high-threat infectious hazards, establishing and maintaining experts’ networks to monitor, detect, understand, and manage emerging or reemerging high-threat infectious diseases in the Region.
The principal risk factors contributing to the emergence and rapid spread of epidemic diseases in the Region include acute and protracted humanitarian emergencies resulting in fragile health systems, increased population mobility (travel and displacement), rapid urbanization, climate change, weak surveillance and limited laboratory diagnostic capacity, and increased human–animal interaction (4).
Members of the genus Orthonairovirus of medical and veterinary significance include Crimean Congo hemorrhagic fever virus (CCHFV) and Nairobi sheep disease virus (NSDV). CCHFV is transmitted by ticks in genera Rhipicephalus and Hyalomma. While neither live virus nor nucleic acid of CCHFV has been detected from bats, serologic evidence suggests past infection of populations of bats across a diverse geographic range. Further, bats are often parasitized by both soft and hard ticks, which occupy a diverse range of ecological niches in endemic countries. A 2016 seroprevalance study by Müller and colleagues examining 16 African bat species (n = 1,135) found that the prevalence of antibodies against CCHFV was much higher in cave-dwelling bats (3.6%–42.9%, depending on species) than foliage-living bats (0.6%–7.1%). They also screened 1,067 serum samples by RT-PCR, but all were negative for CCHFV nucleic acid. Experimental studies to assess the ability of bats to support replication of CCHFV have not been published.
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).
Viruses can be identified by a wide range of techniques, which are mainly based on comparisons with known viruses. Historic methods include electron microscopy, cell culture, inoculation in suckling mice and serology, but these methods have limitations. For example, many viruses cannot be cultivated, excluding the use of cell line isolation and serologic techniques, and can only be characterized by molecular methods. In 2011, Bexfield summarized the different molecular techniques that identify new viruses such as microarray, subtractive hybridization-based and PCR-based methods. Although these techniques have allowed the discovery of many viruses, the prior knowledge of similar viruses is required. Recent advances in sequence-independent PCR-based methods have overcome this limitation, and Sequence-Independent Single Primer Amplification (SISPA), Degenerate Oligonucleotide Primed PCR (DOP-PCR), random PCR and Rolling Circle Amplification (RCA) methods have emerged. The end result of most of these PCR methods is amplified DNA that requires definitive identification by sequencing.
Novel DNA sequencing techniques, known as “Next-Generation Sequencing” (NGS) techniques, are new tools providing high-throughput sequence data with many possible applications in research and diagnostic settings. With the development of different NGS platforms, it is now possible to sequence all viral genomes in a given sample without previous knowledge about their nature with the use of sequence-independent amplification followed by high-throughput sequencing. This combination of techniques, known as viral metagenomics, allows the discovery of completely new viral species within a complex sample and, due to decreasing costs, are nowadays exponentially increasing.
NGS techniques are able to generate a huge number of sequences, ranging from thousands to millions of reads, in only one reaction. In order to fully benefit from this depth of sequencing to identify infectious agents present in a given environment, host DNA/RNA should previously be removed from samples. Preliminary treatments are therefore required prior to nucleic acid amplification and sequencing, mainly based on nucleases treatments and/or viral purification by ultracentrifugation on sucrose, cesium chloride or glycerol gradients. These strategies are known as “Particle-Associated nucleic acid amplification”, i.e., they try to isolate intact (i.e., infectious) viral particles from their environment, protected from the action of nucleases. Subsequent low amount of nucleic acids have required the use of Sequence-Independent Amplifications (SIA) such as SISPA, DOP-PCR, random PCR, RCA. Although these techniques allow generating enough nucleic acid material for sequencing, their main disadvantage remains that they distort quantitative analyzes by introducing bias of amplification in viral diversity studies. As a consequence, quantitative analyses of the composition of resulting viromes may not reflect the reality.
In diagnostic virology, in either human or veterinary medicine, viral metagenomics has allowed the discovery of causative viral agents of disease conditions. Virome analyses have also been conducted to describe the baseline viral diversity in healthy human conditions, as a prior knowledge before studying the viral flora of pathologic conditions.
In the same way, the use of viral metagenomics as a tool for arboviral and zoonotic disease surveillance requires prior knowledge of the viral diversity associated to hematophagous arthropods and animals in close contact with humans. This review thus summarizes our current knowledge of the diversity of viral communities associated with several arthropods, wildlife and domestic animals and present its potential applications for the surveillance of zoonotic and arboviral diseases.