Some patients suffer from maculopathy or retinopathy. Patients noticed the loss of central vision or blurred eye occurring at various times after infection; e.g., from immediately after the disease onset to several weeks or months later. One or both eyes could be affected [33–35], and the affected eyes had macular edema with exudates containing a white mass covering the macular area with or without retinal hemorrhage, vasculitis, infarction or vitreous haze [34–39]. In addition, retinal detachment, uveitis, or arterial occlusion [35,36,39–41] was reported in some patients. In many cases, a complete recovery of vision does not occur, and chorioretinal scarring can remain in macular and paramacular areas, in spite of the resorption of exudates [34,35,37–41], while some patients show partial improvement in vision after several months of RVFV infection.

Maar et al. described a case of encephalitis in a RVF patient. The patient exhibited symptoms of sudden fever, rigor, and retro-orbital headache for two days. He had fever again at the 22nd day after the onset of illness and experienced neck rigidity lasting for five days from the 25th day. Subsequently, he was sometimes confused and otherwise mentally affected, and experienced temporal vision loss without detectable retinopathy. He also exhibited convulsive attacks, hyperflexia and fever until the 50th day. His serum contained anti-RVFV hemagglutination (HAI) antibodies of 1:160 at the 25th day and 1:640 at the 40th day, while his cerebrospinal fluid (CSF) contained 1:2 of HAI antibody at the 28th day and 1:64 at the 50th day. The CSF also contained an increased number of white blood cells consisting mainly of lymphocytes at the 28th day, indicative of the possible occurrence of viral meningitis or meningoencephalitis. The patient recovered after treatment with amantadine, rifampicin, and dexamethasone for two weeks, although the effect of therapy could not be evaluated precisely.

Another case with encephalitis and retinitis was described by Alrajhi et al.. The patient had a fever, ataxic gait, and bilateral retinal hemorrhage. She could not count fingers, and the CSF contained many leukocytes, including lymphocytes. Her consciousness level was decreased. She was discharged on day 30 of the illness to her home, at which time she was awake, blind, quadreparetic, and incontinent. Moreover, her neurologic conditions did not improve for the next year.

An additional report described a patient who had persistent hemiparesis for four months after the onset of illness, and another paper reported 12 RVF patients, who developed neurological signs and symptoms, including meningeal irritation, confusion, stupor and coma, hypersalivation, teeth-grinding, visual hallucinations, locked-in syndrome, and choreiform movement of upper limbs; in these patients, the histopathological lesions in brains were characterized by focal necroses associated with an infiltration of round cells, mostly lymphocytes and macrophages, and perivascular cuffing.

Zika virus is a new emerging mosquito-borne virus belonging to the Flaviviridae family of viruses (9). This family is comprised of 4 genera: Flavivirus, Hepacivirus, Pegivirus, and Pestivirus (10). Zika virus belongs to the Flavivirus genus, which antigenically and phylogenetically is related to the Spondweni virus (9, 11). Many important human pathogens are included in this genus, for instance, Dengue, West Nile, Yellow fever, tick-borne encephalitis, Japanese encephalitis, Murray Valley encephalitis and St. Louis encephalitis viruses. These viruses are associated with a range of infections from asymptomatic or self-limiting febrile infections to some fatal diseases such as hemorrhage, shock, meningitis, and encephalitis (12).

In Flaviviridae family, all members have enveloped viruses with a single-stranded RNA genome of positive polarity (10) which contained one open reading frame (ORF) with two flanking noncoding regions (at 5′ and 3′ end) (13). The genomes are 5′ capped without a 3′ poly (A) tail.

A polyprotein is coded by the ORF then processed into three structural proteins (the envelope (E), the capsid (C) and the precursor of the membrane (prM)) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (1, 12). The ssRNA is held within an icosahedral capsid shaped from 12-kDa protein blocks; the nucleocapsid is surrounded by a host-derived membrane contained two viral glycoproteins (14).

Similar replication strategies are employed by the members of Flaviviridae family despite significant differences in tissue tropism, transmission, and pathogenesis (10).

Viral encephalomyelitis is an important cause of morbidity and mortality worldwide, and many encephalitic viruses are emerging and re-emerging due to changes in virulence, spread to new geographic regions, and adaptation to new hosts and vectors. The term encephalomyelitis refers to inflammation in the brain and spinal cord that results from the immune response to virus infection. In humans, the viruses most commonly identified as causes of viral encephalomyelitis are herpesviruses and RNA viruses in the enterovirus (e.g., polio, enterovirus 71), rhabdovirus (e.g., rabies), alphavirus (e.g., eastern equine, Venezuelan equine, and western equine encephalitis), flavivirus (e.g., West Nile, Japanese encephalitis, Murray Valley, and tick-borne encephalitis), and bunyavirus (e.g., La Crosse) families. Other virus families with members that can cause acute encephalitis are the paramyxoviruses (e.g., Nipah, Hendra) and arenaviruses (e.g., lymphocytic choriomeningitis, Junin). However, this is certainly not a complete list, because for most cases of human viral encephalitis the etiologic agent is not identified, even when heroic attempts are made.

The primary target cells for most encephalitic viruses are neurons, although a few viruses attack cerebrovascular endothelial cells to cause ischemia and stroke or glial cells to cause demyelination, encephalopathy, or dementia–[5]. Widespread infection of neurons may occur or viruses may display preferences for particular types of neurons in specific locations in the central nervous system (CNS). For instance, herpes simplex virus (HSV) type 1 often infects neurons in the hippocampus to cause behavioral changes, while poliovirus preferentially infects motor neurons in the brainstem and spinal cord to cause paralysis and Japanese encephalitis virus infects basal ganglia neurons to cause symptoms similar to those of Parkinson’s disease.

Because infections with encephalitic viruses are initiated outside the CNS (e.g., with an insect bite, skin, respiratory, or gastrointestinal infection), innate and adaptive immune responses are usually mounted rapidly enough to prevent virus entry into the CNS. Therefore, most viruses that can cause encephalitis more often cause asymptomatic infection or a febrile illness without neurologic disease, and encephalomyelitis is an uncommon complication of infection.

West Nile virus (WNV) is an enveloped spherical, single-stranded RNA flavivirus that is transmitted to birds by ornithophilic mosquitoes, mainly belonging to the genus Culex and is occasionally transmitted to mammalian hosts. The geographical area of WNV circulation encompasses most of Africa, Israel, North America and South America, Australia and scattered areas in southern and central Europe, including Russia, Czech Republic, Hungary, Greece, Romania, Italy, Southern France, Portugal, Turkey and Spain. At least two distinct genetic lineages have been identified among the WNV isolates in diverse areas: lineage 1 includes most of the American, European and some African strains, whereas lineage 2 contains mainly sub-Saharan African isolates, although some lineage 2 strains have been detected in humans and mosquitoes outside Africa. Additional lineages have been proposed: lineages 3, 4 and 5 include viruses isolated from Czech Republic (Rabensburg strain), Caucasus and India, respectively. A sixth lineage was recently described in Indonesia and a putative seventh lineage has been identified in Spain.The strains belonging to lineages 1 and 2 have up to 30% nucleotide divergence. This wide diversity together with the elevated threat for human health posed by WNV infections resulted in the development of a variety of methods for laboratory diagnosis. It is important to emphasise that about 80% of the human infections caused by WNV remain asymptomatic and therefore approximately 20% of cases become clinically evident. The clinical syndromes associated with WNV human infections are predominantly mild flu-like fevers (WNV fever) of which less than 1% develops severe neuroinvasive disease. For detail about the case classification of the WNV infection and for proposals concerning laboratory diagnosis of this virus please refer to the conclusion of this paper.

This paper reviews the currently available techniques for the identification of WNV infection in humans. Four External Quality Assessment (EQA) studies have been performed: two for the molecular diagnostics of WNV infections, and two for the serological methods. The results of these studies are presented at the end of this review, focusing on the main diagnostic problems.

Currently, the laboratory methods for the diagnosis of infection by WNV belong to two main categories: serology and viral detection.

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.

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.

Dengue virus (DENV) is a single-stranded, positive-sense RNA virus that belongs to the Flaviviridae family. There are four different serotypes of DENV. DENV is transmitted by Aedes aegypti and Aedes albopictus mosquitoes, and DENV infection causes dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). DSS is fatal in 1%–2.5% of cases with intensive treatment, and the mortality rate increases to >20% in the absence of proper treatment. Thus far, DENV infection has not been reported in South Korea, except in patients who have traveled to endemic areas, such as Asia-Pacific, Central and South America, and Africa, which are the major sited of dengue fever outbreaks. According to the World Health Organization, 40% of the world population lives in high-DENV areas. An estimated 390 million people are infected with DENV, and about 20,000 of them die each year. In 2014, 113 cases of dengue fever were reported in Yoyogi park in central Tokyo. Similarly, in Europe, from 2012 to 2013, more than 3,000 people were infected with DENV in Portuguese Madeira on the Atlantic Ocean. In addition, 2 and 17 cases of dengue fever in persons who had no previous travel abroad were reported in France and Croatia, respectively, which were the first known cases of self-occurrence in Europe since 1928 and suggest the possibility of a future DENV epidemic. A. aegypti and A. albopictus, which spread DENV, mostly inhabit the tropics and subtropics. These mosquitoes also carry several other infectious viruses, such as Chikungunya, West Nile fever, and yellow fever viruses, which have already been imported into Europe and the Americas by cargo ships and airlines. In 2013, subtropical mosquitoes were found in Jeju Island, South Korea. Based on their genome sequence, these mosquitos were identified as a strain of A. albopictus, a species which is known to transmit DENV, from Vietnam. Until now, all cases of DENV infection in Korea have been shown to be imported, and >95% of patients had returned to Korea after being infected in Southeast Asia or South Asia. However, as the domestic climate becomes more subtropical, these subtropical insects can survive much longer, and the possible occurrence of subtropical mosquitoes may result in an influx of DENV in South Korea.

The immunology and pathology of DENV have not yet been elucidated, and this has limited the development of vaccines and effective therapeutics. Since 1940, when the development of DENV vaccines and treatments began, preclinical testing has not provided sufficient evidence for efficacy or accurate toxicity profiles at the clinical trial stage. Dengvaxia, the first DENV vaccine (developed by Sanofi), is a quadrivalent vaccine that was marketed in five countries, including Brazil, beginning in June 2016. However, its efficacy is only about 60%, which is less effective than that of other vaccines for diseases such as measles and poliomyelitis, which are more than 95% effective. Children under the age of 9 years and adults over the age of 45 years, the main victims of dengue fever, are not eligible to receive Dengvaxia due to unexplained side effects. In addition, it has been shown to have insufficient effects on serotype 2 infection due to interference between serotypes. Furthermore, a component of the vaccine, the non-structural protein of the yellow fever virus, induces a T-cell reaction to yellow fever rather than an antibody response to DENV. A clinical trial of about 30,000 people conducted in 10 countries showed that the vaccine may cause serious symptoms in patients. The different clinical outcomes of vaccine administration, such as low efficacy and unexplained side effects, appear to result from the lack of an established disease model for testing the safety and efficacy.

Zika virus RNA is detectable in different types of body fluids such as blood (serum or plasma) (47–50), urine (49, 51), saliva (52) semen (40), breast milk (49), conjunctival fluid (53) and amniotic fluid (54); and brain and placental tissues of congenitally infected fetuses (41, 42).

Reverse transcriptase PCR (RT-PCR) is highly sensitive and specific and known as the “gold standard” for ZIKV diagnosis (55). Regarding Zika virus-specific RT-PCR, several conventional and real-time assays targeting prM, E, NS1, NS3, NS4, and NS5 genes have been developed (50, 56–59). However, to the best of our knowledge, the Food and Drug Administration (FDA) has approved only one commercial assay e.g. Cobas Zika test (Roche) which is a qualitative nucleic acid test for screening Zika virus RNA in blood donors. Additionally, FDA has authorized the use of several molecular assays under an Emergency Use Authorization (EUA) (60) all of them are based on RT-PCR (such as Triplex Real-Time RT-PCR, a multiplex assay for detection of Zika virus, Dengue virus, and Chikungunya virus (CDC) (61), Zika virus RNA qualitative real-time RT-PCR (Quest Diagnostics Infectious Disease, Inc.) and RealStar Zika virus RT-PCR kit (Altona Diagnostics, GmbH)) expect Aptima Zika virus assay based on Transcription-Mediated Amplification (TMA) Technology (60).

Molecular diagnosis of Zika virus infection in human usually performs on plasma or serum specimens within the first week after onset of clinical symptoms (60). Although, there are several lines of evidence for advantage of urine for Zika virus RNA detection because of the long duration of viral shedding in this easily collectable specimen (55

, 12, 62), interesting findings indicating the shorter persistence of ZIKV RNA in urine versus serum have been observed (60, 63, 64).

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

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

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

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

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

DENV is widespread in the temperate and tropical regions of the world, and each year approximately 50–150 million people are infected (Bhatt et al., 2013), with over 10 000 deaths (Stanaway et al., 2016). Symptoms of Dengue fever include a high fever, headache, vomiting, muscle and joint pains, and skin rash. Severe cases of disease is usually associated with secondary infection with heterologous types of DENV (Halstead, 1988), and can develop into Dengue hemorrhagic fever (with hemorrhage, thrombocytopenia and blood plasma leakage), or into Dengue shock syndrome, both of which are potentially fatal (Kularatne, 2015).

Infection of 129Sv/Ev mice with 108 pfu DENV-2 via the intravenous (IV) route resulted in 87% survival (26 out of 30 mice), and inoculation with 4.4×104 pfu DENV-1 via IV resulted in 93% survival (40 out of 43 mice) (Shresta et al., 2004). In their study, Shresta et al. challenged 5–6 week old Ifnar–/– mice (129Sv/Ev background) with DENV-2 strain PL046 (n=12) and DENV-1 strain Mochizuki (n=16) at the same doses and inoculation routes. While no lethality was observed, sera and major organs harvested from infected mice at 3 and 7 dpi showed the presence of virus in all sera, liver, spleen, and lymph node samples, as well as some brain and spinal cord samples (Shresta et al., 2004). Interestingly, mice deficient for the type I and II IFN receptors (AG129) showed uniform death from DENV-2 infection with animals dying between 7–30 dpi, and DENV-1 as well with mice succumbing to disease between 7–14 dpi (Shresta et al., 2004).

A previously well 69-year-old Australian man traveled to Thailand in early May 2017. The planned duration of travel was 12 days, and he did not attend a travel clinic prior to departure. The patient did not take malaria chemoprophylaxis, nor did he have a prior history of Japanese encephalitis virus (JEV) vaccination. He flew to Phuket, before traveling north to the popular tourist destination of Khao Lak, where he stayed in a beachside holiday resort. Heavy rainfall occurred during the trip, which limited holiday activities. He did not travel to rural or remote areas, but did receive numerous mosquito bites. On the eighth day of travel, he became unwell with lethargy and generalized muscle aches. He flew to Bangkok on the ninth day of the trip, and over the following 3 days his symptoms included ongoing lethargy, poor appetite, and drenching sweats, but no headache, meningism, or confusion. He returned to Australia on day 12 of travel and was admitted to a regional hospital the following day, now the fifth day after symptoms commenced (Figure 1).

Shortly after admission, on day 7 of his illness, he became confused. Cerebrospinal fluid (CSF) obtained by lumbar puncture demonstrated a glucose of 3.5 mmol/L (reference interval, 2.2–3.9 mmol/L), protein of 1.3 g/L (reference interval < 0.45), polymorphs of 280 × 10^6/L, lymphocytes of 90 × 10^6/L, and red blood cells of 54 × 10^6/L. He was commenced on empiric broad-spectrum antibiotics with vancomycin, meropenem, benzyl penicillin, and acyclovir. Due to a deteriorating conscious state, he was intubated the following day and transferred to a tertiary center. Neurological examination upon arrival revealed a generalized flaccid paralysis. A magnetic resonance image of the brain demonstrated no abnormalities. Seizure activity developed on day 10 of his illness, for which anticonvulsant medication was commenced.

Diagnostic assays performed on the initial and a repeat CSF (on day 10) were negative using conventional gel-based and real-time multiplex polymerase chain reactions (PCRs) for herpes viruses (herpes simplex 1 and 2 and varicella zoster), enterovirus, pan-flavivirus (Murray Valley encephalitis [MVE], Kunjin, dengue, West Nile, Zika, yellow fever, JEV), respiratory viruses (influenza, respiratory syncytial virus, parainfluenza, human metapneumovirus, picornavirus, adenovirus, coronavirus), meningococcus, and pneumococcus. The conventional pan-flavivirus PCR and JEV real-time PCR performed on plasma were also negative (see the Supplementary Data for detailed methods). HIV and syphilis serology were negative, as were bacterial cultures and screening swabs for Burkholderia pseudomallel.

On the fourteenth day of illness, JEV immunofluorescence serology (Euroimmun) performed in parallel on plasma from day 7, day 12, and day 13 of illness, was positive. The JEV IgG titre increased across the serial bleeds from 160 to 1280 to >2560 (confirmed by neutralization), suggesting recent infection, and JEV IgM was detected in all 3 specimens. Serology performed on CSF from day 10 was also positive for JEV IgM and IgG. The presence of measles IgG in plasma but not CSF was consistent with local production of JEV antibodies in CSF rather than contamination. Serology for MVE (EIA—total antibody) was weakly positive on day 7 but negative on days 12 and 13 and thought to represent nonspecific cross-reactivity. Dengue virus IgM, IgG, and NS1 antigen were not detected.

Given the results of serological testing, serial samples of plasma, whole blood, and urine were tested using the conventional gel based pan-flavivirus PCR and JEV real-time PCR. Notably, both urine and whole blood specimens were found to be persistently positive for JEV (confirmed by sequencing of the NS5 region), while plasma was found to be persistently negative (Figure 1). Reproducibility of these findings was confirmed by re-extracting and retesting the 4 most recent urine and whole blood specimens directly from the primary samples, and repeat testing was performed on separate PCR runs. Whole-genome sequencing and subsequent phylogenetic analysis (Supplementary Figure 1) identified the isolated JEV strain (VIDRL_JEV aligned) as a member of Genotype I. Our patient’s isolate localized within a subclade of viruses isolated from Thailand, geographically aligning with the travel history.

Aliquots of urine and whole blood were inoculated onto cultured cells to assess viral infectivity (see the Supplementary Data for detailed methods). Cytopathic effects were observed 7 days postinoculation from the day 14 urine specimen. The cell culture supernatant was tested by JEV real-time PCR and was found to be positive with a high level of detection, suggesting efficient viral replication.

Japanese encephalitis viral RNA was detected in urine samples out to day 26 after the onset of symptoms and in whole blood up until the final specimen was tested on day 28.

Electromyogram and nerve conduction studies performed on day 22 were consistent with an acute motor-axonal neuropathy or anterior horn cell pathology. No clinical improvement was evident after 4 days of intravenous immunoglobulin (IVIG) administered at a dose of 1 g/kg daily, and the patient died 30 days after the onset of symptoms.

YFV is endemic in tropical areas of Africa and South America (WHO, 2016b), when the virus was introduced via the slave trade during the 17th century. Many infections are symptomatic, but if clinical symptoms appear, they include fever, chills, appetite loss, nausea, muscle pains, and headaches. A small percentage (~15%) of cases will go on to develop more severe disease including jaundice, dark urine, vomiting and abdominal pain. Hemorrhage from the mouth, nose, eyes or stomach may occur and 50% of patients with these symptoms succumb to disease (WHO, 2016b). YFV was responsible for ~127 000 severe infections and 45 000 deaths in 2013 (WHO, 2016b), with increased incidence over the past decades, and the risk of an outbreak in urban centers is a serious public health threat (Barrett & Higgs, 2007).

Inoculation of wild-type 129 mice SC in each rear footpad with 104 pfu of YFV did not result in any weight loss or death (Meier et al., 2009). In their study, Meier et al. (2009) challenged 3–4 week old Ifnar–/– mice (129 background) with YFV strains Asibi or Angola73 under the same conditions. The mice were shown to be susceptible to the challenge, with death occurring between 7–9 dpi. Additionally, the mice developed viscerotropic disease with virus dissemination to the visceral organs, spleen and liver, in which severe damage of the organs can be observed with gross pathological examination and hematoxylin/ eosin staining. Elevated levels of MCP-1 and IL-6 in these organs are suggestive of a cytokine storm (Meier et al., 2009).

Japanese encephalitis (JE) is caused by the Japanese encephalitis virus (JEV), and is one of the most important mosquito-borne diseases with a mortality rate as high as 20% to 50%, and is widely distributed in most of East and South-east Asia and parts of Oceania. Up to 50,000 human cases of JE are reported annually in Asian countries, of which 10,000–15,000 result in fatality. A high proportion (nearly 50%) of survivors, especially young children and those greater than 65 years of age, exhibit permanent neurologic and psychiatric sequelae. A wide range of animals including swine, equines and birds can also be infected. Pigs, as well as birds, serve as amplifying and reservoir hosts,. Further, JEV infection has accounted for significant economic losses in the pig industry due to fetal encephalitis and reproductive failure in pregnant sows and hypospermia in boars. There is no specific treatment available for JE, and vaccination is the only effective way to prevent JEV infection in humans and domestic animals. JEV non-structural protein 1 (NS1) has been shown to induce both humoral and cell-mediated immunity against JE,. Further, like other flaviviruses, NS1 is able to elicit protective immunity without the risk of antibody-dependent enhancement. These characteristics make NS1 an attractive alternative immunogen. As such, much research is currently being devoted to NS1-based vaccine development,,. Although NS1 is not present in the virion, NS1-induced antibodies can protect against infection in vivo by an undetermined mechanism, which presumably depends on the Fc portion of the antibody since they kill their target cells through a complement-dependent pathway,.

JEV is a member of the family Flaviviridae, and the genus Flavivirus, and is primarily transmitted by Culex mosquitoes. Besides JEV, the Japanese encephalitis virus serocomplex of Flaviviridae includes the West Nile virus (WNV), Saint Louis encephalitis virus (SLEV) and Murray Valley encephalitis virus (MVEM). JEV serogroup viruses and Dengue virus (DENV) have a similar ecology; it is very common that two or more of these flaviviruses co-circulate in some regions of the world–[15], and cross-reactivity can be demonstrated among these flaviviruses in serological tests. These cross-reactive responses could confound the interpretation of results during serological testing, including neutralization tests and enzyme-linked immunosorbent assays (ELISA). This serves to emphasize the utility of virus-specific epitopes for the differential diagnosis of disease and epidemiological surveys. The serological cross-reactivity is primarily caused by cross-reactive epitopes on the structural protein E,. In contrast, NS1 is more specific in serological testing of flavivirus infections, and it has been reported that NS1 can induce antibodies without cross-reactivity among flaviviruses, and even among different serotypes of DENV,, therefore the development of an NS1-based specific serological diagnosis is of great interest,,. First, it is necessary to precisely identify the B-cell epitopes on NS1. In this study we have identified and characterized five JEV NS1-specific epitopes with monoclonal antibodies. This work demonstrates progress toward the development of a specific serological diagnostic test for JEV infection, extends our understanding of the antigenic structure of JEV NS1, and could help inform vaccine design.

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.

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.

The Flaviviridae family includes approximately 80 members divided into four genera: Flavivirus, Pestivirus, Pegivirus, and Hepacivirus. The Flavivirus genus can be further divided into four categories: mosquito-borne, tick-borne, no known vector (NKV), and insect-specific (ISF) viruses. Mosquito- and tick-borne flaviviruses, including Dengue (with four serotypes), Japanese encephalitis virus (JEV), Yellow fever virus (YFV), Saint Louis encephalitis virus (SLEV), West Nile virus (WNV), Murray Valley encephalitis virus (MVE), and tick-borne encephalitis virus (TBEV), are important pathogens responsible for human diseases, such as encephalitis, fever, and haemorrhagic fever.

NKV viruses are primarily restricted to bats and rodents. ISF viruses are restricted to mosquitoes, such as Culex and Aedes, and include the Aedes flavivirus (AeFV), Calbertado virus, cell-fusing agent virus (CFAV), Chaoyang virus, Culex flavivirus (CxFV), Culex theileri flavivirus (CTFV), Kamiti River virus (KRV), Lammi virus, Nakiwogo virus (NAKV), Nounane virus, Quang Binh virus (QBV), and Palm Creek virus (PCV) [3–12].

Mosquito- and tick-borne flaviviruses are transmitted to humans through haematophagous insects during blood meal feeding. Viruses obtained from vertebrate host initially replicate in the midgut within 5–7 minutes of exposure. After escaping the midgut, the virus spreads to other tissues via haemolymph and can be transmitted through infected salivary glands and saliva [13–15]. The period from the initial infection in the midgut to when the vector transmits the virus is termed the extrinsic incubation period (EIP), and this time period varies from 7 to 14 days [14, 15]. However, viruses have been detected in the salivary glands of DENV-infected Aedes aegypti as early as 24 hours. Thus, the EIP depends on the virus, the mosquito, and certain environmental factors [14, 15].

Flaviviruses are single stranded-RNA viruses with positive polarity (ssRNA+). These viruses are approximately 11 kb in length, with a single open reading frame (ORF) encoding a polyprotein that is co- and posttranslationally processed through cellular and viral proteases into three structural (C, M, and E) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. The ORF is flanked at the 5′ and 3′ ends by two untranslated regions (5′ and 3′ UTRs) that are important in viral translation and replication.

The virions are spherical and approximately 50 nm in diameter. The capsid (C) protein interacts with the viral genome to form the nucleocapsid, which is surrounded by a lipid bilayer containing the membrane (M) and envelope (E) proteins.

Flavivirus is a genus of the family Flaviviridae that contains a large number of viral agents capable of causing encephalitis and jaundice. Most flaviviruses are arboviruses and transmitted to the human population by a bite from infected mosquitoes or ticks. Flaviviruses typically contain a positive sense single-stranded RNA genome of approximately 10-11kb in length. The genome encodes 3 structural proteins (Capsid, prM, and Envelope) and 8 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5 and NS5B). The viruses are enveloped with a diameter of around 50nm, and appear icosahedral or spherical when observed under the electron microscope. Individual members such as dengue (DENV), yellow-fever virus (YFV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV) and West Nile virus (WNV) cause significant morbidity and mortality worldwide.

DENV is a major public health concern on a global scale with an estimated 400 million infections and 100 million clinical cases in 2010. Most of these patients will carry the disease asymptomatically. However, around 5% of infected individuals will progress to severe dengue, an illness characterized by plasma leakage leading to hypovolemic shock, hemorrhage, and potentially death. The case-fatality rate for individuals with severe dengue can be as high as 10% if untreated, or 0.1% with appropriate clinical management.

Alphaviruses are a diverse group of viruses that are classified as belonging to the group IV Togaviridae family of viruses. There are over thirty members in the alphavirus group that are able to infect a wide range of vertebrates including humans, rodents, fish, birds, and horses. At the genomic level alphaviruses consist of a positive sense, single-stranded RNA genome 11 to 12kb in length with a 5’ cap, and 3’ poly-A tail. Alphavirus particles are enveloped, have a size of around 70 nm in diameter under the electron microscope and appear to be spherical with a 40 nm isometric nucleocapsid. Like flaviviruses the main mode of transmission to the human population is via bites from infected mosquitoes. Notable viruses that infect the human population include chikungunya (CHIKV), Barmah Forest virus (BFV), Mayaro virus (MAYV), O'nyong'nyong virus (ONNV), Ross River virus (RRV), Una virus and Tonate virus.

Epidemics of flavivirus and alphavirus occur globally on an annual basis with different degrees of severity. Table 1 shows a small selection of recent flavivirus/alphavirus outbreaks worldwide.

The global distribution and severity of flavivirus and alphavirus infection requires accurate surveillance tools and timely diagnosis to ensure infected patients obtain the best medical treatment options and alert authorities to possible outbreaks of disease.

The most accurate method to diagnose viral agents is real time Polymerase Chain Reaction (RT-PCR). Primer and probe sequences complementary to the viral RNA are designed and cycled through a series of steps with positive samples seen as amplification curves on a RT-PCR instrument. This process can be completed in less than 1 hour, which significantly assists in patient management.

However, members of the flavivirus and alphavirus families are quite heterogeneous at the RNA level, therefore it can be difficult to design a single set of primers and probe sequences that can detect each of the families at the genus and species level. An example of this is DENV that contains four serotypes, each being quite diverse at the genomic level. Like most current dengue RT-PCR assays, the CDC DENV-1-4 RT-PCR Assay detects serotypes 1–4 using an individual primer pair and probe for each type. Assays that can universally detect all DENV serotypes have been described but these assays still employ more than 2 primers to detect all subtypes.

In order to simplify and improve the detection of alphaviruses and flaviviruses in clinical samples, we developed a commercially available 3base assay that is able to detect the presence of the target alphavirus or flavivirus using a single primer and probe set for each type. 3base assays use chemical modification to reduce the complexity of genomes from 4 to 3 base, which enable screening primers and probes with fewer mismatches to be developed so that bias in amplification efficiency across species is greatly reduced. (Fig 1).

The 3base protocol (Fig 2) deaminates all cytosine residues in nucleic acids to a uracil intermediate. This process makes closely related species more similar at the genomic level. This novel method ultimately means that primers and probe sets can be designed that have fewer mismatches and are able to hybridise to previously heterogeneous target regions with higher efficiency, thus improving PCR amplification of species that contain large numbers of individual pathogens.

The modification process of the genomic nucleic acids to a 3base form does not sacrifice specificity and individual typing primers can be constructed to detect the exact organism responsible for disease.

The method has been used to successfully detect the presence of high risk HPV in clinical samples and the presence of pathogens, including Norovirus, in patients with gastrointestinal disease.

We have utilised the method to produce pan-species assays for the detection of all flavivirus, alphavirus and dengue serotypes 1–4 and successfully applied these assays to screen samples in the 2016/17 Vanuatu dengue outbreak.

The first clinical report of Rift Valley fever (RVF) in humans was made in an area near Lake Naivasha of the Rift Valley province in Kenya in 1930.1 RVF epizootics and epidemics in livestock and humans have periodically occurred and were geographically restricted to sub-Saharan Africa, but since 2000, this disease has spread to the Arabian Peninsula.2 Rift Valley fever virus (RVFV) infection is correlated with several risk factors, including contact with sick animals or contaminated products or exposure to virus-carrying mosquitoes.3 Sero-epidemiology revealed anti-RVFV IgG antibodies among livestock and human in countries such as Djibouti where RVF outbreaks have never been reported in either humans or animals,4 suggesting the presence of subclinical virus circulation in non-epidemic areas.

Although RVFV has been described in an Angolan returning from South Africa,5 and circulating RVFV was reported among animals and humans in other Central African countries such as Central African Republic,6 no epizootic or epidemic occurrences have been reported in Angola. Herein, we describe the first case of imported RVFV infection from Angola to China. The longitudinal observation of the clinical manifestations and pro-inflammatory immune mediators of this severe case were reported, and our phylogenetic analysis revealed the virus to be a novel reassortant between lineages E and A.

Because flaviviral infection persists for the life of the vector, the opportunities for competition or viral interference in the vector are higher than in humans, where the infection is only transient and is cleared through the immune system. The evaluation of viral interference during flavivirus infection is relatively easy to detect in cell lines, and it has primarily been examined in mosquito C6/36 cells (from Aedes albopictus) [12, 23, 30, 31, 35, 39, 41–48], TRA-171 (from Toxorhynchites amboinensis), Sf9 (from Spodoptera frugiperda), C7-10, and U4.4 cells (from Aedes albopictus) [29, 35]. Homologous or heterotypic, but not heterologous, viral interference is frequently observed during superinfections (Table 2), and this condition is particularly evident in persistently infected cells [23, 31, 32, 44, 49].

However, some exceptions have been documented [32, 39, 41, 44]. For example, SINV inhibits DENV replication in C6/36 cells infected 1 hour prior to DENV-4. The same cells persistently infected with Aal DNV and reinfected with DENV-2 showed an important reduction in the severity and mortality of the DENV-2 infection compared with those of noninfected cells, and DENV-2 titres were lower than in naïve cells. Interestingly, C6/36 cells persistently infected with three different viruses, including two flaviviruses, DENV-2 and JEV, result in a stable coinfection with the three viruses without apparent viral interference. These discrepancies indicate that the interference might vary among different flaviviruses and might be influenced through both the type of virus and the cell line used. It has been shown that the interval between the primary and secondary viral infections has an important effect on viral interference. The primary infection of C6/36 cells with either DENV-2 or DENV-4, followed by a secondary infection 1 or 6 hours later with the opposite virus at the same multiplicity of infection (MOI), showed a stronger reduction in the virus titres of the secondary virus when the second infection was performed 6 hours after the first infection. It is likely that DENV-4 infection requires more than 1 hour to establish conditions in which this virus will not be affected by SINV.

Contradictory results have been reported regarding WNV and CxFV infections. Some studies have reported that CxFV-infected C6/36 cells were reinfected with WNV 48 hours later and display significantly reduced titres of the secondary virus at 108 hours postinfection, indicating the presence of homologous viral interference. However, other studies have reported that when the same CxFV-infected cells were reinfected with WNV two days later, homologous viral interference was not observed. These differences could reflect the time of the secondary infections, but more experiments will be necessary to clarify this point.

Flavivirus coinfection experiments have primarily been performed using C6/36 cells [30, 41–43, 46, 48, 50], and recently Aag2 cells have been used; these infections typically result in homologous or heterologous viral interference (Table 3).

Upon admission to our hospital on day 7, the patient's vital signs were normal (temperature, 37 °C; heart rate, 90 beats per min; respiration rate, 20 per min; and blood pressure, 120/70 mm Hg). He was awake, alert and fully oriented. Physical examination revealed scleral icterus, no splenomegaly or hepatomegaly and no joint tenderness or swelling. Rash and haemorrhagic tendency were absent.

The laboratory tests on day 7 showed renal failure and severe liver damage, with creatinine levels of 1005 μmol/L, blood urea nitrogen levels of 35 mmol/L, total bilirubin levels of 83.8 μmol/L, alanine aminotransferase of 5910 IU/L, aspartate amino transferase of 7570 IU/L and prothrombin levels of 74% (Table 1). Additional abnormal laboratory values included lactate dehydrogenase (1880 U/L), creatine kinase (6680 U/L), and myohaemoglobin (1200 ng/mL; Table 1). The patient was transferred to the intensive care unit because of his presentation of multiorgan dysfunction, including acute kidney injury, acute hepatitis, acute myocardial injury, pancreatitis and rhabdomyolysis.

Computed tomography scanning showed pneumonia in the double upper and lower lobes of the lung, pleural effusion, cholecystitis and a small amount of ascites; however, the head CT scan was normal (Figure 1). An ultrasonic cardiogram showed that the left ventricular ejection fraction was 60% and that the heart structure had no obvious abnormalities.

RVFV nucleic acids were detectable in the serum and saliva samples with Ct values of 28.7 and 31.0, respectively, on day 7.

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).

All viruses in the genus Flavivirus (family Flaviviridae) possess a single-stranded, positive-sense RNA genome of approximately 11 kb. The genome usually encodes a single open reading frame (ORF) that is flanked by 5' and 3' untranslated regions (UTRs) of ~100 and ~400–700 nt, respectively. The ORF encodes a large polyprotein that is co- and post-translationally cleaved to generate three structural proteins, designated the capsid (C), premembrane/membrane (prM/M) and envelope (E) proteins, and seven nonstructural (NS) proteins in the gene order: 5'–C–prM(M)–E–NS1–NS2A–NS2B–NS3–NS4A–2K–NS4B–NS5-3'. The genomes of some flaviviruses appear to encode an additional protein as a consequence of ribosomal frameshifting as discussed later in this review.

Despite their similar genomic organizations, flaviviruses possess fundamental differences in their host ranges and transmissibilities. Most recognized flaviviruses are transmitted horizontally between hematophagous arthropods and vertebrate hosts and are therefore considered to be dual-host viruses. Dual-host flaviviruses can be further divided into mosquito/vertebrate and tick/vertebrate viruses. Examples of mosquito/vertebrate flaviviruses include dengue virus (DENV), yellow fever virus, Japanese encephalitis virus (JEV) and West Nile virus (WNV), all of which are human pathogens of global concern. Flaviviruses of localized public health concern include St Louis encephalitis virus (SLEV) and Murray Valley encephalitis virus (MVEV). Tick/vertebrate flaviviruses associated with serious human disease include tick-borne encephalitis virus, Langat virus and Powassan virus. Not all flaviviruses cycle between arthropods and vertebrates; some have a vertebrate-specific host range while others appear to be insect-specific. Vertebrate-specific flaviviruses, also known as No Known Vector (NKV) flaviviruses, can be divided into two groups: those isolated exclusively from rodents (e.g., Modoc virus; MODV) and those isolated exclusively from bats (e.g., Rio Bravo virus). It has been suggested that NKV flaviviruses are maintained in nature by horizontal transmission among hosts. Insect-specific flaviviruses (ISFs) can also be divided into two distinct groups (Figure 1). ISFs in the first group are phylogenetically distinct from all other known flaviviruses and, for the purpose of this review, will be referred to as classical ISFs (cISFs) since they were discovered first. ISFs in the second group phylogenetically affiliate with mosquito/vertebrate flaviviruses and, for the purpose of this review, will be referred to as dual-host affiliated ISFs (dISFs). This group is apparently not monophyletic. There has been a dramatic increase in the number of ISFs discovered in the last ten years and this is partly due to advances in the methods available for virus detection.

In addition to the growing number of ISFs recently described, cISF-like sequences, designated cell silent agent (CSA), have been found integrated in the genomes of Aedes spp. mosquitoes. One sequence was shown to encode a NS1-NS4A-like transcript. Partial E, NS4B and NS5-like sequences were also identified. The occasional integration of flavivirus sequences into the mosquito genome could occur due to the reverse transcriptase and integrase activities of co-infecting or endogenous retroviruses, and indeed the integration of viral sequences into the host genome has since been documented for many other RNA viruses and host species. Most integrated sequences are highly fragmented or have internal stop codons but several encode intact ORFs. Importantly, some genome-integrated sequences may be transcribed and therefore, the detection of flavivirus-like RNA in an organism is not necessarily proof that the organism carries an active flavivirus infection.

The median age of patients was 57.2 years (range, 23–88) and the male-to-female ratio was 1 to 2.27; 219 patients (92.02%) were farmers and 19 (7.98%) were workers or students. Among patients, 52 (21.85%) reported a tick bite within 2 weeks (5–14 days) before the onset of clinical manifestations; the remaining patients did not recall receiving a tick bite.

The main clinical features in confirmed patients included sudden onset of fever (>37.5°C −40°C) lasting up to 10 days, fatigue, anorexia, headache, myalgia, arthralgia, dizziness, enlarged lymph nodes, muscle aches, vomiting and diarrhea, upper abdominal pain, and relative bradycardia (Table 1). A small number of cases suffered more severe complications, including hypotension, mental status alterations, ecchymosis, gastrointestinal hemorrhage, pulmonary hemorrhage, respiratory failure, disseminated intravascular coagulation, multiple organ failure, and/or death. Most patients had a good outcome, but elderly patients and those with underlying diseases, neurological manifestations, coagulopathy, or hyponatremia tended to have a poorer outcome.

Laboratory tests showed that confirmed patients characteristically developed thrombocytopenia, leukopenia, proteinuria, and elevated serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels (Table 2). Biochemical tests revealed generally higher levels of lactate dehydrogenase, creatine kinase, AST and ALT enzymes, especially AST.

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.