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Based on the results from several studies, the WHO has launched a new dengue classification. This classification divides dengue cases into a) cases with/without warning signs and b) severe dengue cases. However, it is important to note that numerous research groups have debated the rational of this classification as it does not fit their unique local settings. The criteria for dengue case classification are presented in Fig. 1.
Clinically, dengue infection has a broad spectrum of features. The vast majority of cases are asymptomatic and passes unnoticed. Typically, the symptoms start to be prominent after an incubation period of 3–10 days. The severity of the clinical manifestations varies from mild symptoms to severe life threatening symptoms in the case of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Predicting the progression of the mild signs to a severe DHF/DSS remains a challenge due to non-specificity of clinical presentation and the incomplete understanding of pathophysiology of the disease and its underlying molecular mechanisms.
The early signs of the disease are non-specific. According to the WHO classification (2009), DF is characterized by febrile episode (≥40 °C for 2–7 days) frequently associated with rash, nausea, vomiting, and headache. Although the disease affects people of all ages from infancy through to adulthood, epidemiological data showed that children tend to tolerate this phase of illness better than adults. The persistence of the aforementioned symptoms and appearance of other symptoms, such as abdominal pain, mucosal bleed, and lethargy and restlessness can be seen 3–7 days later. Laboratory analysis of mild dengue fever cases usually shows abnormal leukocyte counts and moderate elevation of the hepatic amino-transferase enzyme activity. The emergence of these symptoms is a warning sign for disease progression to severe form (DHF/DSS) if therapeutic intervention is not undertaken. At this stage clinical intervention and continuous surveillance are imperative to prevent vascular leakage, especially in an endemic area.
Clinical signs of classical swine fever usually appear 5–10 days after infection (occasionally longer). An individual pig may show one of four types of clinical effect; Peracute (sudden death, especially at the beginning of a farm outbreak), Acute (fever, depression, weakness, anorexia, conjunctivitis, diarrhoea or vomiting, purple discoloration of abdominal skin, or necrosis of the tips of extremities, and neurological signs), Chronic (weight loss, hair loss, dermatitis, discoloration of abdomen or ears) and subclinical. Affected pigs may recover or relapse, depending on the severity of the disease. Reproductive effects is also common; abortions, stillbirths, mummifications and also congenital tremor of piglets.
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.
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.
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.
Chikungunya virus (CHIKV) (family Togaviridae, genus Alphavirus), is the causative agent of chikungunya fever. After first isolation of CHIKV in 1952 in present-day Tanzania, outbreaks and epidemics were limited to regions of Asia, Africa, and the Pacific Islands. In 2013, CHIKV emerged for the first time in the Americas, with sustained autochthonous transmission and rapid spread through the region [1–3]. The acute symptoms of CHIKV infection are similar to those of infection with other arbovirus species, including Dengue virus (DENV), Zika virus (ZIKV), and Mayaro virus (MAYV), each presenting with a constellation of symptoms including fever, headache, and myalgias/arthralgias. Long-term, CHIKV infections have been linked with persistent arthralgias in a subset of cases; it has also been reported that upwards of 90% of CHIKV-infected individuals are symptomatic, in contrast to findings with ZIKV, where it is estimated that only 20% of infected persons are symptomatic.
The similarity of the clinical presentation of acute-phase arbovirus infections is further complicated by the potential for simultaneous infection with multiple arboviruses. In a recent literature review, co-infections with CHIKV and DENV ranged from 1% to 34% of patients. However, virtually no data are available on frequency of co-infection with CHIKV and arboviruses other than DENV. Even where good laboratory diagnostic facilities are available, identification of co-infections often does not occur, as there is a tendency to cease investigation once an initial pathogen has been identified, and/or identification of a second pathogen may require facilities for virus isolation, which may not be available.
As part of ongoing studies of acute undifferentiated febrile illness in a cohort of school children in rural Haiti, we identified 82 children with RT-PCR-confirmed CHIKV infections during May-August 2014, corresponding with the time period when the Caribbean CHIKV epidemic was moving through Haiti. Specimens were also simultaneously screened by RT-PCR for DENV1-4, then additionally for ZIKV. Aliquots of the plasma specimens were then inoculated onto cell cultures for the isolation of additional pathogens of potential interest. We report here results of these studies, focusing on rates of arbovirus co-infection in our patient cohort and potential sources of origin of the co-infecting strains.
An ideal animal model for the study of a human disease is one which utilizes a route of infection that mimics the natural transmission of the pathogen; the ability to obtain disease with an infectious dose equivalent to that causing disease in humans; as well having a disease course, morbidity and mortality similar to that seen with human disease. Additionally, the animal model should have a mode(s) of transmission that mimics human cases. Factors which subsequently allow more detailed inferences about disease pathogenesis include the availability of reagents to evaluate host innate and adaptive immune responses to the pathogen, and histopathological changes in the host which result from infection or the host response to infection. These findings can then be compared to what is known of human disease. The utility of a small animal model of human disease for study of therapeutic efficacy is augmented when large numbers of animals are available for use in appropriately, well-powered studies. Even if all aspects of an animal model of disease are not completely faithful to what is known of human disease, important information regarding therapeutic efficacy can be gleaned from their use in “pre-clinical” studies.
The published literature on clinical manifestations of systemic human orthopoxvirus disease is derived from historic literature descriptions of human smallpox and more recent descriptions of human monkeypox disease. The clinical-descriptive literature on human monkeypox is expected to grow in the next five years, as data acquisition and analysis from an ongoing study in the Democratic Republic of Congo is finalized. Currently available literature is largely derived from WHO-sponsored surveillance efforts in West Africa and the Congo Basin in the 1980s, after the first recognition of human disease in these areas, and subsequent analyses of public health response data and human research studies following the introduction of West African clade virus into the U.S. in 2003. Human monkeypox, as described through the active surveillance and case ascertainment studies sponsored by WHO in the 1980s, was depicted as resembling discrete ordinary smallpox. In natural human infection, exposure leading to infection is believed to occur via a respiratory route, with subsequent progressive viremias/lymphemia, ultimately leading to seeding of the skin to generate a generalized rash. Percutaneous exposure, also leading to generalized rash formation, has also been described for both viral infections. The disease pathogenesis has been conjectured and modeled largely from animal studies; initial models were using ectromelia infection of mice; some kinetic observations of virus shedding and viremia have been made in human studies of smallpox and monkeypox. The time course of disease is generally thought to include an asymptomatic phase of 10–12 days, during which time the virus initially enters the host, replicates, seeds reticuloendothelial organs, replicates, then spreads via the bloodstream (inducing a febrile response) which is the first symptomatic hallmark of disease. The fever is usually described as occurring 10–12 days post initial exposure/infection. The range has been 7–17 days. Fever is accompanied by other symptoms, including headache, backache, myalgias, and or abdominal pain. Two to three days following the fever, rash develops—initially presenting as a macular, then papular, then vesicular and pustular eruption. Scabbing then begins. Each stage of rash lasts 1–2 days. Approximately 2–3 weeks post initial symptoms, scabs begin to separate from the skin. Death and disease severity have had some correlation with rash burden in epidemiologic studies of hospitalized smallpox patients. Severe outcomes are more frequent in unvaccinated, younger age groups; death occurs within the first week of illness in cases with hemorrhagic manifestations, and during the second or third week of illness in “ordinary” cases. In the human monkeypox cases studied in Zaire/DRC between 1981–1986, of the 33 deaths among 338 patients, all occurred in unvaccinated children less than eight years of age. Death occurred during the first week of illness in 21%, the second week in 52%, and the third week of illness in the remaining 27%.
The development of small animal models for the study of monkeypox virus (MPXV) has been quite extensive for the relatively short period of time this pathogen has been known. Initial animal models were designed to address natural history in potential or surrogate reservoir host species, as well as studies of disease in primates. Routes of exposure were designed to evaluate disease if respiratory or percutaneous exposures occurred, or in some cases to simply address whether virus would replicate in the animal model system. Factors that influence the outcome of a challenge study include the age of the animal at time of infection, inoculation route used, and the viral dosage given. Additionally, the strain of MPXV (currently delineated as belonging to Congo Basin clade or West African clade) used in the study may influence the disease severity.
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.
Chikungunya virus (CHIKV) is a mosquito-borne virus belonging to the genus alphavirus, family Togaviridae and spread by Aedes mosquitoes. CHIKV is the aetiological agent of chikungunya fever (CF) and was first isolated in 1952 from the serum of a febrile patient during an outbreak in Tanzania, Africa. CHIKV infection is characterized by an abrupt onset of fever lasting two to five days, frequently accompanied by arthralgia. The disease is usually self-limiting, but joint pain symptoms can persists for weeks up to years [2–4]. Other common symptoms include muscle pain, headache, nausea, fatigue and rash similar to the dengue virus infection. Besides CHIKV, the Alphavirus genus includes viruses such as O'nyong'nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BFV), Sindbis virus (SNV) and Mayaro virus (MAYV).
Since its discovery in Africa, CHIKV has repeatedly caused outbreaks in Africa, India, Southeast Asia, the Middle East and Europe with irregular intervals [6–10]. Phylogenetic analysis of CHIKV showed that the virus has evolved into three distinct genotypes: Asian, West African and Eastern/Central /South African (ECSA). A single-base mutation E1-A226V in a strain of the ECSA genotype enhanced replication of the virus in Ae. albopictus, and led to a large-scale epidemic on La Reunion in 2005. This ECSA genotype was subsequently associated with epidemics in the Indian Ocean region, and the Asian genotype has been associated with recent outbreaks in the Pacific region.
The first local transmission of CF in the Americas was reported from the Caribbean islands in December 2013. The local transmission of the disease has been reported in 45 countries or territories throughout world with more than 1.7 million suspected cases (CCDR October 20, 2015, http://www.cdc.gov/chikungunya/geo/index.html). It appears to have been introduced twice to Brazil, once from the Pacific region and once from Africa.
Current diagnosis of CHIKV is based on three main laboratory methods: virus isolation, reverse transcription-polymerase chain reaction (RT-PCR) and serological tests such as plaque reduction neutralizing test (PRNT), enzyme-linked immunosorbent assays (ELISA) or immunofluorescence test (IFT). Commonly, blood and serum samples are used as specimens for CHIKV diagnosis. Depending on the type of sample and the time of sample collection relative to symptoms (acute or convalescent phase of disease), an appropriate diagnostic method is applied to the samples.
A pronounced viraemia of up to 109 viral genomes can be observed mainly on days 1–6 after onset of disease and in some cases for longer, therefore, virus isolation and RT-PCR are performed on acute phase specimens collected during the first week after onset of symptoms. Several RT-PCR assays have been published for the detection of CHIKV RNA in clinical specimens and mosquito samples [17–23]. Real-time RT-PCR based assays are suitable for clinical diagnosis due to the closed tube assay format, the option for quantification of viral load, high sensitivity and specificity. Serological tests are applied to either acute or convalescent phase samples for the detection of IgM and IgG anti-CHIKV antibodies. Serological diagnosis is confirmed by direct detection of IgM anti-CHIKV antibodies or by determining a four-fold increase in CHIKV-specific antibody titers in acute and convalescent samples by ELISA, IFT or PRNT tests.
Since the clinical picture of CHIKV is similar to that of DENV and Zika virus, a simple and rapid molecular assay would be needed to select the best treatment approach. The recombinase polymerase amplification (RPA) assay utilizes enzymes and proteins in order to allow the amplification of the DNA at a constant temperature (38–42°C). The presence of the amplicon is detected via the exo-probe, which include an internal abasic site mimic (tetrahydrofuran, THF) flanking fluorophore and Quencher as well as pathogen-specific 30 and 15 bases at both the 5´ and 3´ prime ends, respectively. Upon the hybridization of the exo-probe to the complementary sequence the Exonuclease III cleaves at the THF site and the fluorescence signal is generated. RPA assay was successfully developed for the detection of DENV and YFV as well as other human and veterinary pathogens [25–32]. Moreover, a mobile suitcase laboratory was established for allowing the deployment of the RPA in the field for identifying Ebola virus infected case.
In this study, we developed and evaluated a reverse-transcription recombinase polymerase amplification (RT-RPA) assay as potential point-of-care (POC) diagnostic tool for rapid detection of CHIKV. The RT-RPA assay was designed by targeting the non-structure protein 1 (nsP1). The sensitivity and specificity of the method was evaluated using strains of the three genotypes of CHIKV and compared to reference RT-PCR methods. Finally, the performance of the RT-RPA assay was evaluated on acute-phase serum samples for clinical diagnosis of CHIK.
Border disease is an important disease in sheep, caused by infection of the foetus in early pregnancy. Surviving lambs are persistently viremic, and the virus is present in their excretions and secretions, including semen. Ruminants and possibly also pigs can be readily infected by contact with these persistent excretors or with acutely infected sheep. Acute infections in immunocompetent animals usually are transient and subclinical and result in immunity to challenge with homologous but not heterologous strains of virus. The disease is characterised by low birth weight and viability, poor conformation, tremor, and an excessively hairy birth coat in normally smooth-coated breeds. Kids may also be affected, and a similar condition occasionally occurs in calves. The disease has been recognized in most sheep-rearing areas of the world.
Although viruses infecting humans had already been described since 1901 and viruses were suspected to play a role in diarrhea, it lasted until 1972, when the first virus causing gastroenteritis (norovirus) was identified in an outbreak of diarrhea in Norwalk (California, United States). Shortly after the discovery of norovirus several other viruses causing gastroenteritis were discovered: rotavirus in epithelial cells of children with gastroenteritis, astrovirus in infantile diarrhea cases, enteric adenoviruses in the feces of children with acute diarrhea, and sapovirus during an outbreak of gastroenteritis in an orphanage in Sapporo, Japan. All these viruses spread via the fecal-oral route through person-to-person transmission and are described in more detail below.
Noroviruses are part of the family Caliciviridae and outbreaks of norovirus gastroenteritis have been reported in cruise ships, health care settings, schools, and in the military, but norovirus is also responsible for around 60% of all sporadic diarrhea cases (diarrhea cases where an enteropathogen could be found), reviewed in the literature. The pathogenesis of norovirus infection has been tested in vivo. Filtrated norovirus was given to healthy volunteers after which most of them developed diarrhea. Culturing of the virus, however, has been a problem since its discovery, yet one study has recently described the cultivation of norovirus in B cells, and has revealed that co-factors, such as histo-blood antigen expressing enteric bacteria, are probably needed before enteric viruses can be cultured in vitro. Sapoviruses are also members of the Caliciviridae. There are five human genogroups of sapovirus described which account for 2.2%–12.7% of all gastroenteritis cases around the globe. Sapovirus outbreaks occur throughout the year and can be foodborne. For sapoviruses it has been described that the virus was not found before onset of an outbreak, and that it was found in 95% of the patients during an outbreak, while it declined to 50% after an outbreak, indicating that the virus introduces disease in a naturally infected host.
Rotavirus infection is the most common cause of viral gastroenteritis among children; however, parents of infected children also often become ill and as a result rotavirus is the second most common cause of gastroenteritis in adults. Studies in human volunteers have shown that infection with rotavirus causes diarrhea, results in shedding of the virus and a rise in antibody anti-virus titer after infection. Additionally, astroviruses infections are common, accounting for about 10% of all sporadic diarrhea cases. Astrovirus has been isolated from diseased people, filtrated and administered to healthy individuals after which in some of the volunteers diarrheal disease was observed and astrovirus was shed in their stools. The virus can replicate in human embryonic kidney cells and was detected by electron microscopy (EM). Adenoviruses are responsible for around 1.5%–5.4% of the diarrhea cases in children under the age of 2 years, reviewed in the literature. Of the 57 identified adenovirus types, only adenoviruses type 40 and 41 are associated with diarrhea. Next to these two types, adenovirus type 52 can also cause gastroenteritis, although it has been argued whether type 52 is actually a separate type since there is not sufficient distance to adenovirus type 41. Adenoviruses can generally be propagated in cell lines; however, enteric adenovirus 40/41 are difficult to culture, reviewed in the literature.
Three main clinical syndromes can be distinguished after hantavirus infection, i.e. hemorrhagic fever with renal syndrome (HFRS) mainly caused by Seoul, Puumala and Dobrava viruses; nephropathia epidemica, a mild form of HFRS caused by Puumala virus; and hantavirus cardiopulmonary syndrome, which may be caused by Andes virus and Sin Nombre virus. There is no curative treatment for hantavirus infection, and eliminating or minimising contact with rodents is the best way to prevent infection. The case fatality rate of HFRS varies from <1% to 12% depending on the virus type. Over 50 000 nephropathia epidemica cases with Puumala virus infections have been registered in Europe, and even over 175 000 in Western Russia. Approximately 150,000 to 200,000 humans are hospitalized each year because of hantavirus infections worldwide. European hantaviruses of the family Bunyaviridae cause HFRS. Infection in humans occurs through inhalation of aerosolised virus particles from excreta of chronically infected wild rodents. Puumala is reported throughout most of Europe (excluding the Mediterranean region) wheras Dobrava, carried by the yellow-necked mouse (Apodemus flavicollis) and Saaremaa, carried by the striped field mouse (Apodemus agrarius), are reported mainly in eastern and central Europe. In Germany, several hantaviruses pathogenic for humans are circulating, i.e. Puumala virus, Dobrava virus and Tula virus. Puumala virus is carried by the bank vole (Clethrionomys glareolus) and leads mostly to a mild form of disease.
The course of HFRS is highly variable, ranging from frequently asymptomatic to a lethal outcome. Host genetic factors influence the clinical outcome. The most common symptoms are high fever, headache, abdominal pain, backache and nausea or vomiting. Proteinuria, haematuria and acute kidney injury are signs of renal involvement. Classically, HFRS occurs in five distinct phases: febrile, hypotensive, oliguric, polyuric and convalescent. A severe course of disease comprises oliguria, high blood creatinine and a high leukocyte count, the disease severity depends on the hantavirus genotype. A minority of patients needs transient dialysis treatment, but complete recovery is the usual outcome. Outinen et al. defined the severity of hantavirus infection by serum creatinine and thrombocytes count. The role of immune response and cytokine expression during Puumala virus infection has been described previously. Techniques for the identification of novel hantaviruses by specific cell culture models had been established. Only few cases of Puumala virus infection with severe disease had been reported for Germany recently.
In the year 2012, the highest number of human hantavirus infections were observed and more than 2800 cases were reported, 30% of these infections were located in Southwestern Germany. Case numbers started to rise earlier during the year than had been reported in previous epidemics in 2007 and 2010, and are the largest ever reported in this region. The early rise might be associated with a birch mast year in 2011, followed by an early and massive reproduction of the reservoir of bank vole populations during winter 2011 and spring 2012. However, a possible influence of climate change on the survival, emergence and epidemiology of hantaviruses is difficult to predict.
It has been suggested that viral load and immunological factors including cytokines are involved in the pathogenesis of Puumala virus infection. The extent and level of viremia depends largely on the hantavirus type, viral load is considerably lower in HFRS caused by Puumala virus when compared to other hantaviruses. Thus viral load clearly plays a role in disease pathogenesis. In a recent study, patients infected with Dobrava virus were found to have a higher viral load than Puumala infected patients. The aim of this study was to analyse TGF-ß serum levels in patients with hantavirus infection and to evaluate the level of TGF-ß in mild and severe course of disease.
Waterbirds and shorebirds of the orders Anseriformes (mainly ducks, geese and swans) and Charadriiformes (mainly gulls, terns and waders) are considered the natural host reservoirs of LPAI viruses (see Fig. 1). In wild birds LPAI viruses predominantly infect epithelial cells of the intestinal tract, and are subsequently excreted in the faeces. However, infection of wild birds with LPAI viruses is typically sub-clinical and occurs in the absence of obvious lesions,,. Every year, LPAI viruses cause outbreaks amongst waterbirds. These outbreaks are most commonly associated with the increased presence of juvenile, immunologically naïve birds in the population and occur during migration when contact rates between, and within, populations are high. The relatively high virus prevalence in waterbirds may be due, in part, to virus transmission through the faecal–oral route via surface waters.
On admission, all study patients were seropositive for anti-Puumala IgG and IgM antibodies. Out of 77 patients with acute Puumala virus infection, 52 (68%) were male. A seasonal distribution of hantavirus infection was detected in our cohort and for nationwide cases with a peak in summer 2012. Age distribution showed a higher percentage of patients in the age groups of 20–39 (48.1% vs 33.1%; p < 0.05) years and a lower percentage of patients in the age groups of 40–59 (32.5% vs 48.0%; p < 0.05) years in our cohort compared to nationwide data (Figure 1). In contrast to the highest incidence in the age group of 40–49 (28.1% vs 15.6%; p < 0.05) years nationwide, our study patients demonstrated the highest proportion in the age group of 30–39 years (24.7% vs 18.0%; p > 0.05, not significant).
For all patients mean hospitalization time was 4.9 days. On admission, demographic data and clinical symptoms such as fever, myalgia, and headache were similar in patients with mild and severe disease (Table 1). However, patients with a severe course of disease defined by a glomerular filtration rate (GFR) of <30 ml/min had significantly longer hospital stay (6.2 vs 3.6 days) and significantly higher proteinuria levels (1.3 vs 0.6 g/l) compared to patients with mild disease, respectively. The most common symptoms reported on admission were myalgia, fever, headache, shivering, low back pain, and arthralgia (Table 1). All patients had impaired renal function to some degree on admission with an increase in retention parameters over the course of disease, six patients (8%) required intermittent hemodialysis.
Influenza A viruses (Family Orthomyxoviridae) impose a large burden on both human and animal health worldwide. Influenza A viruses can be categorised into different subtypes based on genetic and antigenic differences in the two surface glycoproteins of the virus, the haemagglutinin (HA) and neuraminidase (NA). Wild waterfowl and shorebirds are the natural reservoirs of influenza A virus and can be infected with viruses harbouring combinations of 16 different HA subtypes and nine different NA subtypes. Recently, two novel influenza A virus subtypes (H17N10 and H18N11) have been identified in rectal swabs collected from the little yellow-shouldered bat [Sturnira lilium] and the flat-faced fruit-eating bat [Artibeus jamaicensis planirostris],,. Influenza viruses of this subtype have not been isolated from any other animal order and it is unknown whether these viruses might be able to cross the species barrier. In contrast, there is significant inter-species transmission of influenza viruses from waterbirds, such that animals ranging from domestic poultry to humans can also become infected. Accordingly, infection with influenza virus has wide-reaching ramifications. For example, whilst some influenza virus strains are largely asymptomatic in chickens (and are hence referred to as low pathogenic avian influenza [LPAI] viruses) others cause severe disease in chickens that is often fatal within 48 h (and are hence referred to as highly pathogenic avian influenza [HPAI] viruses). Outbreaks of HPAI viruses can cause devastation for the poultry industry resulting in the mass slaughter of millions of birds. Similarly, outbreaks of influenza viruses amongst thoroughbred horses have disrupted numerous race meetings and resulted in the death of infected horses. In humans, seasonal influenza viruses are a significant cause of morbidity and mortality and constitute an economic burden of $10.4 billion dollars per year in the U.S.A. alone. The diversity and complexity of influenza virus infections across so many different animal species suggests that a one-health approach is the only comprehensive way to reduce the burden of disease. Here, we seek to highlight how influenza viruses spread from their natural avian hosts to mammals, and what the virus needs to overcome in order to ensure the success of these inter-species transmission events. We highlight the consequences that this inter-species transmission has, not only for human health, but also for the health of wild animals and the success of industries such as poultry farming.
Alkhurma hemorrhagic fever virus (AHFV) in humans was discovered in 1994. The first case reported in a butcher from the city of Alkhurma, a district south of Jeddah in Saudi Arabia, died of hemorrhagic fever after slaughtering a sheep. The viral infection has a reported fatality rate of up to 25%. Interestingly, one of the previous reports regarding this disease showed a misunderstanding of the real name of this infection, called Alkhurma, not Alkhumra. Because subsequent cases were diagnosed in patients from the small town known as Alkhurma in Jeddah from where the virus got its scientific name; the name was accepted by the International Committee on Taxonomy of Viruses. Thus, based on evidence, the first case was confirmed to be the butcher, following the slaughtered sheep. Therefore, a study was conducted among affected patients to address this disease as a public health issue. Blood samples were collected from household contacts of patients with laboratory-confirmed virus for follow-up testing by enzyme-linked immunosorbent serologic assay (ELISA) for AHFV-specific immunoglobulin (Ig) G. Samples from persons seeking medical care were tested by ELISA for AHFV-specific IgM and IgG using AHFV antigen. Viral-specific sequence was performed by reverse transcription PCR (TiBMolbiol, LightMix kit; Roche Applied Science, Basel, Switzerland). A total of 11 cases were identified through persons seeking medical care, whose illnesses met the case definition for AHFV, and another 17 cases were identified through follow-up testing of household contacts.
Subsequently, the virus was isolated from six other butchers of different ages (between 24 and 39 years) from the city of Jeddah, with two deaths. The diagnosis was established from their blood sample tests. The serological tests later confirmed four other patients with the disease. From 2001 to 2003, the study on the virus initial identification in the city of Alkhurma again identified 37 other suspected cases; with laboratory confirmation of the disease in 20 (~55%) of them. Among the 20, 11 (55%) had hemorrhagic manifestations and 5 (25%) died. The virus was later identified in three other locations: from the Western Province of Saudi Arabia (Ornithodoros savignyi and Hyalomma dromedarii were found by reverse transcription in ticks) and from samples collected from camels in Najran. AHFV virus was considered as one of the zoonotic diseases; however, the mode of transmission is not yet clear. Recently, it was suggested that the disease reservoir hosts may include both camels and sheep. The virus might also be transmitted as a result of skin wounds contaminated with the blood or body fluids of an infected sheep; through the bite of an infected tick, and through drinking of unpasteurized or contaminated milk from camels.
In humans, this zoonotic disease may present with clinical features ranging from subclinical or asymptomatic features to severe complications. It is related to Kyasanur Forest disease virus, which is localized in Karnataka, India. However, epidemiologic findings suggest another wider geographic location for the disease in western (including Jeddah and Makkah) and southern (Najran) parts of Saudi Arabia, and the virus infections mostly occur in humans. A study was conducted by Alzahrani et al. in the southern part of Saudi Arabia particularly in the city of Najran (with populations of ~250,000), an agricultural city in Saudi Arabia, where domestic animals are reared at the backyard of owners. After the initial virus identification, from January 2006 through April 2009, 28 persons with positive serologic test results were identified. Infections were suspected if a patient had an acute febrile illness for at least two days; when all other causes of fever have been ruled out. Additionally, data analysis indicated that patients infected with the virus were either in contact with their domestic animals, involved in slaughtering of the animals, handling of meat products, drinking of unpasteurized milk, and/or were bitten by ticks or mosquitoes. Symptoms consistent with AHFV infection—including fever, bleeding, rash, urine, color change of the feces, gum bleeding, or neurologic signs—then develop. Fortunately, infected patients responded to supportive care (including intravenous fluid administration and antimicrobial drugs when indicated), with no fatal cases.
In summary, AHFV is a zoonotic disease with clinical features ranging from subclinical or asymptomatic features to severe complications. Another study highlighted different characteristics of the exposure to the blood or tissue of infected animals in the transmission of AHFV to humans. Of the 233 patients confirmed with infections, 42% were butchers, shepherds, and abattoir workers, or were involved in the livestock industry. More recently, a study on infection using C57BL/6J mice cells showed that the clinical symptoms of the disease were similar to the presentations in humans. However, Alkhurma disease resulted in meningoencephalitis and death in Wistar rats, when high titers to the infection occurred. In addition, exposures to mosquito bites are regarded as potential sources of transmissions of the infection; however, very few available data support this. Although, available data shows that Alkhurma virus has been isolated following mosquito bites. However, another study suggested that mosquitoes may play a role only as a vector in the transmission of the disease.
In the 1980s and 1990s some viral agents were identified for which the direct association with disease is less clear. Aichi viruses are members of the Picornaviridae identified in fecal samples of patients with gastroenteritis. Aichi virus infection has been shown to elicit an immune response. Since their discovery, two case-control studies were performed, but, although both studies only found Aichi virus in stools of diarrheic patients, the prevalence of Aichi virus (0.5% and 1.8%) was too low to find a significant association with diarrhea. In immuno-compromised hosts the virus is found in higher quantities and is not associated with diarrhea. Toroviruses, part of the Coronaviridae, were first identified in 1984 in stools of children and adults with gastroenteritis. Torovirus infection is associated with diarrhea and is more frequently observed in immuno-compromised patients and in nosocomial infected individuals. Retrospective analysis of nosocomial viral gastroenteritis in a pediatric hospital revealed that in 67% of the cases torovirus could be detected. However, only a limited number of studies report the detection of torovirus and therefore the true pathogenesis and prevalence of this virus remains elusive. Picobirnaviruses belong to the Picobirnaviridae and were first detected in the feces of children with gastroenteritis. Since the initial discovery, the virus has been detected in fecal samples of several animal species, and it has been shown that the viruses are genetically highly diverse without a clear species clustering, reviewed in the literature. This high sequence diversity has also been observed within particular outbreaks of gastroenteritis, limiting the likelihood that picobirnaviruses are actually causing outbreaks, as no distinct single source of infection can be identified.
All 285 patients with FTLS were from the Henan Province of China and were provisionally diagnosed as suspected HGA on the basis of similar clinical manifestations,. They represented four different epidemiologically linked sporadic cases and a few clusters of cases including 79 patients in 2007, seven patients in 2008, 47 patients in 2009, and 152 patients in 2010. The patients presented mainly between April and October, peaking in April-May during the tea-picking season in Henan. All patients resided in mountainous and hilly rural areas. In our study, 238 of 285 patients tested positive for novel bunyavirus infection by RT-PCR and/or IFA.
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–. 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.
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.
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.
Transmission appears to occur more readily if the recipient is immunocompromised or has another comorbidity, such as diabetes. In the largest outbreak described thus far, 23 patients with laboratory-confirmed infection were followed in Al-Ahsa governorate in Saudi Arabia (3). Diabetes mellitus (74%), end-stage renal disease (52%), and lung disease (43%) were underlying illnesses in these patients. Transmission to family members and health care workers was documented in 1 to 2% of contacts, again demonstrating preferential infection of individuals with substantial comorbidities. Unlike another human respiratory coronavirus, the one that caused the severe acute respiratory syndrome (SARS) in 2002–2003, MERS-CoV has not preferentially infected health care workers. At present, it is not known if patients are able to transmit virus before the development of symptomatic respiratory disease. If this does occur, control of a large outbreak will be more difficult. The SARS epidemic was contained, in part, because the majority of patients were infectious only after they developed pneumonia.
One year after MERS first came to light, all cases have been found to have a Middle East connection, with approximately 70% occurring in Saudi Arabia. Within Saudi Arabia, the first cases were recognized in the Al-Ahsa governorate, an area located in the eastern part of Saudi Arabia, which remains the epicenter of the outbreak. Cases have been identified in the United Kingdom, France, Germany, Italy, and Tunisia, in all instances in patients with a history of recent travel to the Middle East (Fig. 1). Global travel is extremely common, so it may be just a matter of time before MERS-CoV cases are identified on all continents. The Centers for Disease Control and Prevention has published definitions of confirmed and probable cases, which provides a uniform approach to evaluating patients with suspected disease (http://www.cdc.gov/coronavirus/mers/case-def.html).
One important caveat is that we do not know the extent of the infection within the wider community. Since most identified patients have underlying diseases, it is possible that MERS-CoV is a common infection, at least in Saudi Arabia, and that patients without significant comorbidities develop a mild respiratory disease or remain asymptomatic. Careful epidemiological studies, which are required to address this question, are hampered by the lack of validated diagnostic tools. Knowing the number of infected humans will provide critical information about the prevalence of MERS-CoV and about the likelihood of developing severe disease.
Very few topics in Virology relate so closely to the general concept of biological complexity as the emergence and re-emergence of viral disease. In the introduction to their classic book, Solé and Goodwin define the sciences of complexity as “the study of those systems in which there is no simple and predictable relationship between levels, between the properties of parts and of wholes”. The emergence of viral disease involves several levels of complexity. The underlying level stems from the population structure of viral populations as they replicate in their standard hosts. Model studies of plaque-to-plaque transfers (bottleneck passages) of foot-and-mouth disease virus (FMDV) in BHK-21 cells (Fig. 1) showed that the pattern of fitness decay of the virus followed a Weibull distribution. This type of statistical distribution suggests that the mutations fixed in the viral genome at each transfer produced a cascade of perturbations in the virus-host interactions that were sensed in the form of a change in virus yield. The unpredictability of the effect of mutations is further reinforced by the increasing evidence that viral proteins are multi-functional so that mutations can alter one or more of the interactions between viral and host components that determine the viral yield per cell.
The second level of complexity results from a network of environmental, ecologic, and sociologic influences that affect the probability that a potentially pathogenic virus comes into contact with a new host. A good number of such influences are subjected to indetermination. To give a specific example, a change in temperature and humidity in a large geographical area may alter flora and fauna, and, as a result, the distribution of viral vectors (arthropods, birds, mammals). However, which vectors and how they will be affected by weather or climate conditions are usually difficult to predict since they depend on sets of ecological interactions. Finally, another level of complexity intervenes in the epidemiological outcome of a viral disease, once it has emerged. Severe acute respiratory syndrome (SARS) faded months after its emergence, while Acquired Immune Deficiency Syndrome (AIDS) has become a severe pandemics, to last for many decades or forever. The difference can be attributed to the pattern of virus-host interaction and the capacity of the virus to be transmitted from an infected host individual into a susceptible individual. Again, these processes fall into the domain of biological complexity. Advances in molecular virology and the development of computational tools for viral surveillance have greatly improved our knowledge of the factors that underlie the emergence and re-emergence of viral disease, and, interestingly and paradoxically, we understand some of the reasons behind the unpredictability of emergences.
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.