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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.
West Nile virus (WNV) is a zoonotic Flavivirus belonging to the family Flaviviridae. The virus is transmitted by mosquitoes and causes fatal encephalitis in human, equines and birds.
WNV was first isolated and identified in 1937 from a woman presented with mild febrile illness in the Nile district of Uganda. It has been described in Africa, Europe, South Asia, Oceania and North America. Countries with incidence/serological evidence are presented in Fig. (1). In North America, more than 1.8 million people have been infected, with over 12,852 reported cases of encephalitis or meningitis and 1,308 deaths from 1999 to 2010. The mortality rate in human varies from 3-15% and can reach up to 50% in clinically affected horses. WNV in India has been confirmed by seroprevalence and by virus isolation on different occasions from mosquitoes bat and man [42, 45-47]. WNV infection has also been reported in animals and birds.
Horses and human are the main hosts. Animals other than horses may be susceptible to WNV, but rarely become ill. Antibodies have been found in serum samples from bats, horse, dogs, cats, racoons, opossums, squirrels, domestic rabbits, eastern striped skunks, cows, sheep, deer and pigs. The virus is transmitted to humans by mosquitoes. About 20% of the infected people develop fever with other symptoms. Fatal, neurologic illness occurs in less than 1% of infected people.
WNV is amplified by continuous transmission cycles between mosquitoes and birds. Generally Culex mosquitoes are the vectors and passerine birds are the vertebrate reservoirs in enzootic transmission cycles. The virus is carried in the salivary glands of infected mosquitoes and transmitted to susceptible birds during blood-sucking. Competent bird reservoirs sustain an infectious viraemia for 1 to 4 days subsequent to exposure, and then develop life-long immunity. Horses, human and most other mammals rarely develop the infectious levels of viraemia and are dead-end hosts. Few cases in human have been spread through blood transfusions, organ transplants, breast feeding and during pregnancy. The ticks observed to be infected naturally include Ornithodoros maritimus, Argas hermanni and Hyalomma marginatum and the virus has also been isolated from other species of hard ticks in Africa, Europe and Asia [36, 63, 64]. Swallow bugs (Oeciacus hirundinis) have been implicated as vectors in Austria. In most of the horses bitten by carrier mosquitoes, there is no disease. Approximately 33% of the infected horses develop severe disease and die or are affected severely. The time between the bite of an infected mosquito and appearance of clinical signs ranges from 3 to 14 days. The symptoms in horses may vary from none to trembling, skin twitching and ataxia. There can be sleepiness, dullness, listlessness, facial paralysis, difficulty in urination and defecation, and inability to rise. In some horses, there can be mild fever, blindness, seizures, and other signs.
There is no effective treatment for clinical WNV infection in humans, horses or any other animal. Vaccines are available for control of WNV in horses in the USA. Vaccination of horses protects valuable animals from a potentially fatal disease, but trade and competition practices make this undesirable as some countries use positive antibody tests and impose import restrictions. WNV encephalitis is so rare in human that vaccine development may not be feasible economically.
OHFV distribution is restricted to western Siberia (Figure 1). The main vector of OHFV is the meadow tick, Dermacentor reticulatus, which can also transmit the virus to humans. However, humans are mainly infected after contact with infected muskrats (Ondatra zibethicus) which are very sensitive to the infection and often succumb to the infection. Muskrats develop high viremia which can last for several weeks. Human infection occurs through contact with urine, feces, and blood. Secretion of OHFV in unpasteurized goat milk has been reported but no milk-borne outbreaks have been observed. The exact number of annual cases are uncertain because of misdiagnoses and unreported cases, but 165 cases were reported between 1988 and 1997. OHFV may cause a biphasic disease; the initial phase is characterized by high fever, bleeding from the nose, mouth, and uterus. Thirty to fifty percent of the cases experience a second phase characterized by high fever and reappearance of the symptoms from the initial phase. Case fatality rates range from 0.5 to 2.5%. No antiviral treatments are available against OHFV, instead treatment is focused on supportive care to minimize hemorrhage and other complications.
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).
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.
POWV is found in Russia and North America, and is the only TBFV present in America (Figure 1). It is transmitted by Ixodes scapularis, Ixodes cookei, and several other Ixodes tick species, to small and medium size mammals, whereas humans are accidental dead-end hosts. Milk-borne POWV transmission might also be possible since POWV virus has been found to be secreted in milk under experimental settings.
Although not much is known about POWV pathogenesis, recent studies in mice have found that tick saliva was important to enhance POWV transmission and the outcome of disease. Furthermore, it has been demonstrated that POWV infects macrophages and fibroblasts in the skin, shortly after the tick bite, also, other unidentified cells were shown to be infected. Interestingly, macrophages were found to be the primary target for POWV in the spleen, and in the CNS, which is the main target site for POWV infection, neurons have been shown to be the primary target for POWV in mice and humans.
During the last 10 years there has been an increase of POWV in the USA with approximately 100 reported cases. The recent rise in incidence could be due to increased surveillance and diagnosis of POWV, or it may represent a true emergence of the disease in endemic areas, or both. The incubation period ranges from 1 week to 1 month. The symptoms of POWV infection may include fever, headache, vomiting, weakness, confusion, seizures, and memory loss with a case fatality rate of 10%. Approximately half of the survivors experience permanent neurological symptoms, such as recurrent headaches, muscle wasting, and memory problems (https://www. CDC.gov). There are no antiviral treatments or vaccines available against POWV.
Neurologic symptoms of ADEM commonly appear 4 to 13 days after the infection or vaccination1-3). Fever, headache, vomiting, and meningismus are often seen at the time of initial presentation and may persist during hospitalization2,18). Encephalopathy is a characteristic feature and may progress rapidly in association with multifocal neurologic deficits4). Progression of initial neurologic signs to maximum deficits usually occurs within 4 to 7 days1,3). The level of consciousness ranges from subtle lethargy to frank coma. The altered mental status often raises concern regarding the risk of seizures, although these occur in only one-third of patients3,19).
In addition to encephalopathy, the most common neurologic features of ADEM include long tract (pyramidal tract) signs, acute hemiparesis, cerebellar ataxia, cranial neuropathies, including optic neuritis, and spinal cord dysfunction (transverse myelitis)2-4,18,19). Symptoms of optic neuritis include vision loss, pain with eye movement, and an afferent pupillary defect. Inflammation of the optic disc may be seen by direct funduscopic examination if there is extensive involvement of the optic nerve. The imaging of the optic nerve with a gadolinium-enhanced MRI of the brain and orbits is a more sensitive means to diagnose optic neuritis in these patients. Symptoms of transverse myelitis include flaccid paralysis of the legs with a change in sensory level on examination. Bowel and bladder involvement secondary to spinal cord disease results in constipation and urinary retention. The arms can be involved if the demyelinating lesion affects the cervical cord. Respiratory failure may appear with high cervical lesions that extend into the brainstem. Aphasia, movement disorders, and sensory deficits are unusual. The severe phase of ADEM generally lasts from 2 to 4 weeks. Children may show deterioration in their condition after hospital admission, and most of them develop new neurologic signs. Patients usually recover completely from the acute illness, although some have neurologic sequelae.
MS is a clinical diagnosis and is characterized by spatially and temporally distinct recurrent episodes of demyelination in the CNS. Acute inflammation and demyelination in a critical area of the brain, optic nerves, or spinal cord can induce a corresponding neurologic deficit. There are no clinical signs that are unique to this disorder, but some are highly characteristic (Table 1)20). Common symptoms of MS are listed in Table 220). A European observational study of 394 children with pediatric-onset MS and 1,775 patients with adult-onset MS showed that children were more likely than adults to present with isolated optic neuritis, an isolated brainstem syndrome, or symptoms of encephalopathy17).
A clinically isolated syndrome (CIS) is defined as a single monosymptomatic attack compatible with MS, such as optic neuritis. An episode of CIS can produce diagnostic and therapeutic dilemmas (Fig. 1), since the vast majority of children will not have a recurrence after a single demyelinating event of the CNS. The value of examinations, including brain MRI, cerebrospinal fluid (CSF) analyses, and other laboratory studies to identify those at high risk of recurrence, is unclear21).
Eastern equine encephalitis (EEE) commonly called triple E or, sleeping sickness is a rare but serious viral disease affecting horses and man. The disease is transmitted through mosquitoes and man and horses are dead-end hosts.
EEEV belongs to the genus Alphavirus of the family Togaviridae. It is closely related to Venezuelan equine encephalitis (VEE) virus and Western equine encephalitis (WEE) virus. This virus has North American and South American variants. The North American variant is more pathogenic. EEE is capable of infecting a wide range of animals including mammals, birds, reptiles and amphibians. The virus has been reported to cause disease in poultry, game birds and ratites. The disease has also been reported to occur in cattle, sheep, pigs, deer, and dogs though sporadically. The disease is present in North, Central and South America and the Caribbean. EEE was first recognized in the USA in 1831 from an outbreak where 75 horses died of encephalitic illness and EEE virus (EEEV) was first isolated from infection horse brain in 1933. The serological evidence and outbreaks of the disease have also been reported from horses in Canada and Brazil [119, 120]. Countries with incidence/serological evidence are presented in Fig. (3). EEEV infection in horses is often fatal. The human cases were identified first time in 1938 in the north-eastern United States. Thirty children died of encephalitis in this outbreak. The fatality rate in humans was 35%. The outbreaks of the disease also occurred in horses simultaneously in the same regions. A total of 19 human cases of the disease were reported in children between 1970-2010 in Massachusetts and New Hampshire. As per the CDC reports 220 confirmed human cases of the disease occurred in the U.S. from 1964 to 2004. In 2007, a citizen of Livingston, West Lothian, Scotland became the first European victim of this disease after infected with EEEV from New Hampshire. EEE has been diagnosed in Canada, the United States of America (USA), the Caribbean Islands and Mexico [122, 123]. Eighteen cases of Eastern equine encephalomyelitis occurred in six Brazilian states between 2005 and 2009.
Alternate infection of birds and mosquitoes maintains these viruses in nature. Culiseta melanura and Cs. morsitans species are primarily involved. Transmission of EEEV to mammals occurs via other mosquitoes which are primarily mammalian feeders and called as bridge vectors. Infected mammals do not circulate enough viruses in their blood to infect additional mosquitoes. The virus is introduced by mosquitoes, but feather picking and cannibalism also contribute towards the transmission of the disease within the flocks. Most people bitten by an infected mosquito do not develop any symptoms. The symptoms generally appear 3 to 10 days after the bite of an infected mosquito. The clinically affected patients may have pyrexia, muscle pains, headache, photophobia, and seizures. EEEV is one of the potential biological weapons. The disease in horses is characterized by fever, anorexia, and severe depression. Symptoms appear one to three weeks post-infection, and begin with a fever that may be as high as 106ºF. The fever usually lasts for 24–48 hours. In severe cases, the disease in horses progresses to hyper-excitability, blindness, ataxia, severe mental depression, recumbency, convulsions, and death. The nervous symptoms may appear due to brain lesions. This may be followed by paralysis, causing the horse to have difficulty raising its head. The horses usually suffer complete paralysis and die two to four days after symptoms appear. Mortality rates among horses range from 70 to 90%.
There is no cure for EEE. Severe illnesses are treated by supportive therapy consisting of corticosteroids, anticonvulsants, intravenous fluids, tracheal intubation, and antipyretics. Vaccines containing killed virus are used for prevention of the disease. These vaccinations are usually given as combination vaccines, most commonly with WEE, VEE, and tetanus. Elimination of mosquito breeding sites and use of insect repellents may help in control of the disease.
Acute disseminated encephalomyelitis (ADEM) is an immune mediated disease of the central nervous system (CNS) that produces multiple inflammatory lesions in the brain and spinal cord, particularly in the white matter. ADEM should be distinguished from other central inflammatory demyelinating disorders of children, including multiple sclerosis (MS) and clinically isolated syndromes that include optic neuritis, transverse myelitis, and neuromyelitis optica (Devic's disease). Most of these disorders are thought to be caused by immune system dysregulation triggered by an infectious or other environmental agent in a genetically susceptible host.
ADEM is often preceded by a viral or bacterial infection, usually in the form of a nonspecific upper respiratory infection. In 3 previous studies, an antecedent infection was identified in 72 to 77 percent of ADEM patients1-3). In general, patients will present within 1 month of this initial illness. Numerous causative pathogens have been identified to date. Viruses that have been implicated include coronavirus, coxsackie virus, cytomegalovirus, Epstein-Barr virus, herpes simplex virus, hepatitis A virus, human immunodeficiency virus, influenza virus, measles virus, rubella virus, varicella zoster virus, and West Nile virus4-11). Other organisms associated include Borrelia burgdorferi, Chlamydia, Leptospira, Mycoplasma pneumoniae, Rickettsia , and beta-hemolytic Streptococcus4,12).
Less than 5 percent of all ADEM cases follow immunization. Postvaccinal ADEM has been associated with immunization for rabies, hepatitis B, influenza, Japanese B encephalitis, diphtheria/ pertussis/tetanus, measles, mumps, rubella, pneumococcus, polio, smallpox, and varicella12). No infectious agent is isolated in most cases. Currently, measles, mumps, and rubella vaccination are most often associated with postvaccination ADEM. It is important to recognize the significant difference between the incidence of ADEM associated with the live measles vaccination (1 to 2 per million) and the incidence of ADEM previously associated with the measles virus infection (1 in 1,000)13).
The pathogenesis of ADEM is incompletely understood. The proposed mechanism of ADEM is that myelin autoantigens, such as myelin basic protein, proteolipid protein, and myelin oligodendrocyte protein, share antigenic determinants with those of an infecting pathogen12). This model is supported by studies of lymphocytes in children with ADEM. In 1 report, the frequency of T cell reactivity to myelin basic protein was 10 times higher in patients with ADEM than in those with encephalitis or normal controls5).
MS is usually thought to be a disease of young adults. However, pediatric MS, defined as onset of MS before the age of 16, is being increasingly recognized. MS presents before the age of 16 in approximately 5 percent of patients14,15). In less than 1 percent of patients, the onset of MS occurs before the age of 10 years16). In addition, pediatric MS affects girls more than boys, with a female to male ratio of 2.817). Since pediatric MS is rare, a child with recurrent episodes of acute neurologic symptoms and white matter lesions on magnetic resonance imaging (MRI) might be originally misdiagnosed with one of several other disorders, including leukodystrophies, vasculopathies, lymphoma, mitochondrial defects, and other metabolic disorders, rather than MS.
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.
The global discovery of lyssaviruses is of continued scientific interest and is of importance to both public and animal health. Lyssaviruses are known to cause fatal encephalitis, referred to as rabies. The term rabies has induced terror throughout human history, as the rabies virus (RABV) is the only viral pathogen that is associated with 100% fatality following the onset of the clinical disease. Whilst rabies is predominantly circulating within domestic and feral dog populations globally, the presence of lyssaviruses in bats is well established. Historically, rabies’ association with hematophagous bats (Desmodus sp., although primarily Desmodus rotundus) across the Caribbean, and Central and South America, has both embedded a fear of rabies into human populations, as well as driven an irrational and unjustified fear of bats across many cultures. Certainly, bat transmitted human RABV is rare, although in areas where terrestrial rabies has been eliminated, bat rabies remains a constant threat, as exemplified by continued human cases of bat rabies across North America. In endemic areas, human infection with dog rabies results in thousands of human deaths annually. The estimates of human infection are thought to be conservative, because of inadequate diagnostic and reporting systems across Africa and Asia. Wildlife species can also play an important role in the epidemiology of disease, although the paucity of data on wild animal populations, their distribution, and the generally sporadic interactions between different wildlife populations and domesticated carnivore species means that the role of wildlife and the epidemiology of the virus is often unclear. Still, the transmission of the virus between wildlife and domestic terrestrial carnivores is multidirectional, with incursions of domestic dog rabies into fox populations being reported.
The severity of disease caused by lyssaviruses means that the potential for cross species transmission events (CSTs) is of significance to human and animal populations. For the rabies virus, spill over events are considered as those that result in dead-end infection, whilst CSTs result in the sustained onward transmission of the virus in the new host. Spill over from bats species appears common for RABV in the Americas, whilst events involving the other lyssaviruses across the Old World appear to be rare. Whilst spill over events for lyssaviruses have been reported, host switching events are far rarer and have only been described for RABV in the Americas. The factors involved in CSTs with the sustained onward transmission of the virus remain undefined, and endeavours to identify specific amino acid substitutions facilitating virus adaptation to new host species have been, on the most part, unsuccessful. Kuzmin et al. (2012) observed that for sustainability within a bat population, a Serine at position 242 in the viral G protein appeared to predominate, and that contrastingly, an Alanine/Threonine substitution at position 242 appears to facilitate RABV sustainability within the carnivore population. Intensive characterisation of the genetics within viral populations, including quasispecies, may elucidate the molecular mechanisms that facilitate lyssavirus adaption, however opportunities to genetically characterise such events are rare.
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).
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).
The incubation time for CHIKV is relatively short, requiring only 2–6 d with symptoms usually appearing 4–7 d post-infection. Vazeille et al. detected CHIKV in the salivary glands of Ae. albopictus only 2 d after infection. Upon infection, CHIKF tends to present itself in two phases. The first stage is acute, while the second stage, experienced by most but not all, is persistent, causing disabling polyarthritis. Characteristics of the acute phase include an abrupt onset of fever, arthralgia, and in some cases, maculopapular rash,. The acute phase causes such intense joint and muscular pain that makes movement very difficult and prostrates its victims,.
Ninety-five percent of infected adults are symptomatic after infection, and of these, most become disabled for weeks to months as a result of decreased dexterity, loss of mobility, and delayed reaction. Eighteen months after disease onset, 40% of patients are found to still have anti-CHIKV IgM,,,. The chronic stage of CHIKF is characterized by polyarthralgia that can last from weeks to years beyond the acute stage. CHIKV has been shown to attack fibroblasts, explaining the involvement of muscles, joints, and skin connective tissues. The high number of nociceptive nerve endings found within the joints and muscle connective tissues can explain pain associated with CHIKF,.
More than 50% of patients who suffer from severe CHIKF are over 65 y old, and more than 33% of them die. Most adults who suffer from severe CHIKF have underlying medical conditions,,. The other group that is disproportionately affected by severe CHIKV is children. Other complications associated with CHIKV, from most common to least common, include respiratory failure, cardiovascular decompensation, meningoencephalitis, severe acute hepatitis, severe cutaneous effects, other central nervous system problems, and kidney failure,,,,,,.
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 are an important reservoirs of different pathogenic agents, and many of them have already caused disease outbreaks worldwide.More than 200 viruses have been associated with bats, and almost all are RNA viruses probably owing to their great ability to adapt to changing environmental conditions through a higher genetic variability.Bacteria in bats and their putative threat to humans remain poorly studied.
Viral encephalitis is defined as pathological inflammation of the brain parenchyma secondary to viral infection (1). This syndrome is a rare but clinically serious outcome of infection with a range of DNA and RNA viruses. Encephalitis is associated with appreciable mortality and high rates of permanent neurological impairment in survivors, and in most cases there is no available antiviral therapy (1). Annual healthcare expenditure associated with the acute care of patients with encephalitis was estimated in the region of $2 billion (2), although indirect costs are likely much higher. Furthermore, several emerging causes of viral encephalitis are considered by WHO to be a significant threat to global public health (3). Fundamental to addressing this unmet medical need is research to better understand the pathogenesis of viral encephalitis (4).
Evidence of PARV4 infection is most frequently detected by an ELISA for specific IgG antibody to VP-2. This response appears to be sustained over time, as with other parvovirus infections; weak or transient VP-2 IgM positivity has also been reported in acute infection.
PARV4 DNA may be isolated from plasma in acute infection, generally with low viral loads (e.g. ≤3×104 copies/ml),, although acute viraemia of up to 1010 copies/ml has been reported. Asymptomatic viraemia was reported in 8% of children in a Ghanaian cohort. Different studies have reported the duration of viraemia lasting from 30 days up to a mean of 7 months. However, recrudescence or reinfection could also explain these relatively prolonged durations of viraemia. Despite these reports of isolation of PARV4 DNA from serum,,, this is generally uncommon, suggesting that immune containment is good even in immunocompromised hosts.
Flaviviruses, a group of small, enveloped, positive-sense, single-stranded RNA viruses, include several medically very important pathogens. Especially Japanese encephalitis virus, yellow fever virus, West Nile virus, dengue virus, Murray Valley encephalitis virus and tick-borne encephalitis virus (TBEV) are responsible for large outbreaks of fatal encephalitis or hemorrhagic fevers at diverse geographical regions around the world. Tick-borne encephalitis (TBE), a disease caused by TBEV, represents one of the most important and serious neuroinfections in Europe and northeastern Asia. More than 13,000 clinical cases of TBE, including numerous deaths, are reported annually. Despite the medical importance of this disease, some crucial steps in the development of encephalitis remain poorly understood. In humans, TBEV may produce a variety of clinical symptoms, from an asymptomatic disease (70-90% of cases) to a fever and acute or chronic progressive encephalitis. This is influenced by a variety of factors, e.g., the inoculation dose and virulence of the virus, the age, sex and immune status of the host, and also susceptibility based on the host’s genetic background. Studies of animal models and epidemiological studies in humans have shown that many apparently non-hereditary diseases, including infectious diseases, develop predominantly in genetically predisposed individuals and that this predisposition is caused by multiple genes. In humans, a functional Toll-like receptor 3 gene may be a risk factor for TBEV infection. A deletion within the chemokine receptor CCR5 (CCR5Δ32), which plays an important role in leukocyte transmigration across the blood–brain barrier, is significantly more frequent in patients with TBE than in TBE-naïve patients with aseptic meningitis. Moreover, the severity and outcome of TBE is associated with variability in the 2'-5'-oligoadenylate synthetase gene cluster (family members are interferon-induced antiviral proteins that play an important role in the endogenous antiviral pathway) and with the rs2287886 single nucleotide polymorphism located in the promoter region of the human CD209 gene. This gene encodes dendritic cell-specific ICAM3-grabbing nonintegrin (DC-SIGN), a C-type lectin pathogen-recognition receptor expressed on the surface of dendritic cells and some types of macrophages. Taken together, polymorphism in various genes may largely influence the sensitivity of the host to the infection and determine the severity of this disease.
While in humans involvement of genetic factors in the control of the susceptibility to TBEV infection is quite difficult to investigate, mice provide a useful small animal model for such a kind of study. Mice are suitable animal models of infection with TBEV because they can reproduce symptoms and physiopathological markers as observed in severe cases in humans. A high susceptibility of most laboratory mouse strains to flavivirus infection has been genetically mapped to a stop codon mutation in the coding region of the 2´-5´-oligoadenylate synthetase gene Oas 1b.
In our study, we developed an animal model of TBE showing several manifestations of the disease in relation to the genetic background, which is not based on the previously published mutation in the Oas 1b gene. We analyzed the sensitivity to TBEV in CcS/Dem (CcS) recombinant congenic (RC) strains of mice derived from the background strain BALB/cHeA (BALB/c) and the donor strain STS. Each CcS strain contains a unique random set of about 12.5% genes from the donor strain STS and 87.5% genes from the background strain BALB/c. This system has been very useful in research of bacterial and parasitic diseases, as well as in cancer.
In this study, we identified mouse strains that exhibit high, intermediate and low sensitivity to TBEV infection. Virus growth, key cytokine and chemokine mRNA production in the brain and neutralizing antibody response were measured. Our data suggest that the genetic control represents one of the important factors that influence the clinical course of TBE and that also other genes than the previously described Oas 1b are involved in the determination of host susceptibility to the infection. While high neutralizing antibody response might be crucial for preventing host fatality, high local expression of various proinflammatory cytokines/chemokines in the brain during TBE can be associated with a more severe course of the infection and higher fatality. Our data may be instrumental in the development of future therapeutic strategies aimed at treating or preventing TBE neuropathogenesis.
In the recipients of allo-HSCT, the difference in the reported incidence is due in part to asymptomatic or subclinical manifestations in most of viral infections and the changing epidemiology of viruses as well as differences in diagnostic methods. Till now, large-sampled epidemiological data on overall incidence of viral infections are absent in the recipients of allo-HSCT. The limited data show that community acquired respiratory viruses (CARVs) and herpesviruses are the most common pathogens. Among the causes of CARVs respiratory tract infections, a preponderance of respiratory syncytial virus (RSV) and parainfluenza virus (PIV) are reported, followed by influenza virus and human metapneumovirus (HMPV). In herpesvirus family, the incidence of herpes simplex virus (HSV) and varicella zoster virus (VZV) infections as well as cytomegalovirus (CMV) diseases have significantly decreased because of the effective prophylaxis. The reports on human herpes virus (HHV)-6 diseases are increasing in allo-HSCT recipients.
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).
In the recipients of allo-HSCT, most viral infections are opportunistic and closely related with immune status. Thus, factors influencing engraftment and immune reconstitution all potentially impact viral infections. Peripheral blood stem cell transplantation is associated with fewer viral infections than bone marrow and cord blood transplantation due to better hematopoietic and immune reconstitution. Compared with HLA-match related transplantation, HLA-mismatch related and unrelated transplantation have an increasing risk of viral infections because immune reconstitution is delayed by the intensified GVHD prophylactic strategy, such as the use of ATG. GVHD may delay immune reconstitution and is considered an independent risk factor of viral infections. In addition, other factors, such as the serologic status of donors and recipients before transplantation as well as the age of recipients, may also affect the incidence of viral infections. For example, CMV-seronegative recipients receiving graft from CMV-seropositive donors are at high risk of CMV diseases. Children are high-risk population of CARVs infections.
There is currently no definitive clinical syndrome associated with PARV4 infection, and the potential pathogenicity of related hokoviruses in animals is also unknown. In the majority of instances, PARV4 viraemia appears to be self-limiting and asymptomatic, and there is no consistent association with increased severity of co-existing blood-borne viruses.
However, in a minority of reports, a range of possible disease outcomes are described in individuals with evidence of past or current PARV4 infection, including respiratory or gastrointestinal symptoms, hepatitis, rash, and encephalitis (Table 1). Notably, most of these studies describe small numbers of patients, and none is definitively able to attribute clinical manifestations to the presence of PARV4. Establishing cause and effect is further confounded by the close relationship between PARV4 and other blood-borne viruses; for example, although a statistical correlation has been described between PARV4 positivity and early features of AIDS, this association is potentially confounded by the close relationship between PARV4 and both HCV status and individuals with a history of IDU.
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.