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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.
Vesicular stomatitis is a viral disease which primarily affects cattle, horses, and swine. It occurs in enzootic and epizootic forms in the tropical and subtropical areas. The disease is rarely life-threatening but can have a significant financial impact on the horse industry. Vesicular stomatitis virus (VSV) is the prototype of the genus Vesiculovirus in family Rhabdoviridae. The virus has two serologically distinct serotypes, VSV-New Jersey (NJ) and VSV-Indiana (IND). The neutralizing antibodies generated by these two serotypes are not cross-reactive. The IND serogroup has three subtypes IND-1 (classical IND) IND-2 (cocal virus) and IND-3 (alagoas virus) The virus is endemic in South America, Central America, Southern Mexico, Venezuela, Colombia, Ecuador and Peru but the disease has been reported in South Africa in 1886 and 1897 and France in years 1915 and 1917.
The disease has been reported across continents in Belize, Bolivia, Brazil, Colombia, Costa Rica, Ecuador, El Salvador, Guatemala, Honduras, Mexico, Nicaragua, Pakistan, Panama, Peru, USA and Venezuela [91, 92]. Outbreaks historically occurred in all regions of the USA but have been limited to western states in 1995, 1997, 1998, 2004, 2005, 2006, 2009, 2010, and 2012 [93, 94]. While VS has been reported in horses at about 800 premises in eight states. VSV spread to Europe during the First World War and periodically appears in South Africa. The Chandipura virus, a Vesiculovirus caused encephalitis outbreaks in different states of India leading to mortalities in children. Isfahan another virus in this genus is endemic in Iran [89, 97]. The countries with incidence/serological evidence of vesicular stomatitis are presented in Fig. (2).
Clinical disease has been observed in cattle, horses, pigs and camels whereas sheep, goats and llamas tend to be resistant. White-tailed deer and numerous species of small mammals in the tropics are considered as wild hosts. Many species, including cervids, nonhuman primates, rodents, birds, dogs, antelope, and bats have shown serological evidence of infection. Experimentally different animals like mice, rats, guinea-pig, deer, raccoons, bobcats, and monkeys can be infected.
The virus is zoonotic and causes flu-like symptoms characterized by fever, chills, nausea, vomiting, headache, retrobulbar pain, myalgia, sub-sternal pain, malaise, pharyngitis, conjunctivitis, and lymphadenitis in humans. Vesicular lesions may be present in the pharynx, buccal mucosa, or tongue. Encephalitis is rare but may occur in children [107, 108].
The transmission is more likely by trans-cutaneous or transmucosal route. The virus can be transmitted through direct contact with infected animals having lesions of the disease or by blood-feeding insects. In endemic areas, Lutzomyia sp. (sand fly) is proven biologic vectors. Black flies (Simulidae) are the most likely biologic insect vector in USA. Other insects may also act as mechanical vectors. Saliva, exudates and epithelium from open vesicles are sources of virus. Plants and soil are also suspected as the source of virus.
Horses of all ages appear equally susceptible but lesions do not appear in all susceptible horses. The lesions of the disease resemble foot-and-mouth disease in cattle and the other viral vesicular diseases in pigs. The horses are resistant to foot and mouth disease and susceptible to VS. VSV is the only viral vesicular disease of livestock that infects horses. VSV is also the most important of these four viruses as a zoonotic agent for humans. When vesicular stomatitis occurs in horses, blanched raised or broken vesicles or blister-like lesions develop on the tongue, mouth lining, nose and lips. In some cases, lesions also develop on the udder or sheath or the coronary bands of horses. Animals may become anorectic, lethargic and have pyrexia. One of the most obvious clinical signs is drooling of saliva or frothing at the mouth. The rupture of the blisters creates painful ulcers in the mouth. The surface of the tongue may slough. Excessive salivation is often mistaken as a dental problem or colic. There may be weight loss due to mouth ulcers as animal finds it too painful to eat. The lesions around the coronary band may cause lameness and laminitis. In severe cases, the lesions on the coronary band may cause the hoof to slough. Animals usually recover completely within two weeks. Morbidity rates vary between 5 and 70% but mortality is rare. Vesicular stomatitis like disease disabled 4000 horses during the Civil War in 1862. Major epidemics in the US occurred in 1889, 1906, 1916, 1926, 1937, 1949, 1963, 1982, and 1995, with minor outbreaks during many other years. No specific treatment is available for the disease. Anti-inflammatory medications as supportive care help to minimize swelling and pain. Dressing the lesions with mild antiseptics may help avoid secondary bacterial infections. If fever, swelling, inflammation or pus develops around the sores, treatment with antibiotics may be required. The animals should be quarantined at least for 21 days after recovery of the last case before moving to other places. Vaccines for livestock are available in some Latin American countries.
Whereas in Europe and North America most of the influenza cases are reported between December and March, in tropical and subtropical regions such as in Brazil or in Hong Kong cases are seen throughout the year. Epidemic peaks in the tropical areas mostly occur in between those found in the Northern and Southern hemispheres. A recent survey over 7 years in Brazil showed that annual peaks of influenza cases occurred in association with the rainy seasons. Important reports on spatial and temporal data that describe the global circulation of influenza highlight the fact that there is virtually no data from Africa,. Indeed, until recently, the burden of influenza in Africa was believed to be negligible. However, sporadic reports from the Gambia, Senegal, Congo, Madagascar, Kenya, Ivory Coast, and from Gabon, have indicated that influenza is circulating and may be causing epidemics regularly. The study in Gabon recorded extremely high levels of antibodies to influenza A H3N2 virus in schoolchildren. The haemagglutination inhibition (HI) antibodies to this influenza A virus at titers of 1,530 (ranging from 80 to 17,920) indicated that the virus had been circulating within the community in the recent past. In addition, almost all children, had anti-H1N1 HI titers above 40, while 40% showed antibodies to influenza B with HI titers of 40 or above, again highlighting the fact that multiple influenza virus strains are present in the region. The recent swine flu pandemic provides an interesting example. In the WHO influenza A (H1N1 swine flu) update of May 2009, many countries, but none in Africa, reported virus victims; whereas two reports appeared in October 2009 that showed data on confirmed swine flu cases from South Africa and Kenya, indicating that the virus was circulating in Africa, but because of the lack of a rigorous surveillance system, it was not reported as readily.
Plant-made vaccines against influenza viruses are perhaps the poster children for molecular farming: many candidate vaccines made in plants have shown efficacy in animal models; candidate pandemic virus vaccines have been made to the scale of 10 million doses in less than a month, vaccines suitable for outbreak viruses similarly (see review9). Efficacy to homologous challenge has been shown in mice, ferrets and chickens; so too has efficacy to heterologous challenge with high pathogenicity avian influenza (HPAI) strain H5N1 in chickens.10 While most of this work is directed toward protecting humans against potentially pandemic influenza viruses, it is often overlooked that the same vaccine candidates could be equally useful in birds and in swine: indeed, breaking the chain of recycling of influenza viruses that seems to occur in intensively farmed pigs is a prime goal of One Health.11 Other targets for plant-made influenza vaccines include dogs12 and potentially horses.
Our group investigated the potential for making influenza pandemic rapid response vaccines in South Africa by making influenzavirus A/Vietnam/1204/04 (H5N1) haemagglutinin by transient expression in N. benthamiana:13 our success opened up the possibility of making H5 HA as a reagent and potentially as a vaccine, by means hitherto not available in Africa. We went on to use the HA2 portion of the protein as a virus-like particle (VLP)-based display vehicle in plant manufacture for the highly conserved M2e ectopic epitope as an elicitor of broadly neutralising antibodies to all influenzavirus A strains, as a candidate universal vaccine for humans and animals.14
The Ebola virus (EBOV) is an enveloped, negative-strand RNA virus belonging to the family filoviridae in the order of mononegavirale. Four of the five ebolavirus species, Zaire (ZEBOV), Sudan, Tai Forest, and the recently discovered Bundibugyo ebolavirus, are endemic in continental Africa and cause a severe form of viral hemorrhagic fever with high mortality in humans and non-human primates. Reston ebolavirus (REBOV) is sporadic in the Philippines and has caused several epizootics in cynomolgus macaques. REBOV was first isolated in 1989 from cynomolgus macaques imported from the Philippines for medical research in the United States. About 1,000 monkeys died or were euthanized in a quarantine facility in Reston, Virginia. Subsequently, 21 animal handlers at the Philippine exporter and four employees of the quarantine facility were found to have antibodies to the virus, indicating that they had been infected. Epizootics in monkeys in the Philippines were then reported in 1992 and 1996, and all the epizootics have been traced back to a single monkey facility, in Calamba, Laguna in the Philippines. Since the closure of the facility in 1997, no REBOV epizootics in cynomolgus monkeys have been reported.
In October 2008, REBOV infection was confirmed for the first time in swine associated with multiple epizootics of respiratory and abortion-related diseases in the Philippines. In several pools of swine samples collected from geographically distant swine farms, co-infection with REBOV and porcine reproductive and respiratory syndrome virus (PRRSV) was confirmed. Serological studies of limited scale on 13 swine sera in the affected farms failed to detect REBOV antibodies in ELISA, although PRRSV antibodies were detected. It is still unclear how REBOV was spread among swine during the epizootic. Moreover, it is not clear if REBOV infection in the swine population is either sporadic and incidental or common in the Philippines. To try to answer these questions, we prepared multiple serodiagnosis systems for detecting REBOV infection in swine and analyzed swine sera obtained from the affected farms and from farms not associated with any epizootics in the Philippines. The results showed a high prevalence of REBOV infection in swine in the affected farms at the epizootics in 2008; however, REBOV antibodies were not detected in the swine population not associated with the epizootics, indicating that REBOV infection in swine in the Philippines is not common, at least in some parts of Tarlac.
Influenza viruses comprise 4 types: A, B, C, and D. Influenza A viruses are further classified into subtypes on the basis of the characteristics of the 2 main surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), and numbered accordingly. While only 2 influenza A virus subtypes (H3N2 and H1N1pdm09) are circulating among humans, the natural reservoir for nearly all influenza A viruses is wild waterfowl. Of the 18 HA and 11 NA influenza A virus subtypes, all but H17N10 and H18N11 viruses have been identified in birds. Some influenza A viruses circulate among pigs, and others have been identified in a wide number of animal species.
Only influenza A viruses cause seasonal influenza epidemics and rare pandemics among people. Influenza B viruses can cause seasonal epidemics, influenza C viruses typically cause mild respiratory illness and do not cause epidemics, and influenza D viruses primarily affect cattle and are not known to cause illness in people.9
Novel influenza A viruses refer to viruses of animal origin that have infected humans and that are antigenically and genetically distinct from seasonal influenza A viruses circulating among people. We closely monitor novel influenza A viruses, because influenza A viruses continue to evolve and because zoonotic transmission could herald an increasing pandemic influenza health threat. If a novel influenza A virus acquires the ability for sustained human-to-human transmission, a pandemic can result. Accordingly, early detection of pandemic-potential viruses may aid in the control and possible prevention of the next pandemic. Although a swine-origin influenza A virus caused the 2009 H1N1 pandemic,10,11 and sporadic transmission of swine-origin influenza A viruses to humans (termed “variant viruses”) continue to be detected in the United States and in other countries with appropriate laboratory capacity, the public health threat posed by avian influenza A viruses appears to be higher because of their diversity and wide circulation among birds worldwide; the birds' migratory flyways may also be conducive to spread of influenza A viruses. Furthermore, some previous pandemic influenza A viruses have been partly of avian origin.11
The virus is responsible for causing severe and rapidly progressing illness in humans with the respiratory system as well as the central nervous system (CNS) mainly getting affected (Hossain et al. 2008). The signs and symptoms of the disease appear 3–14 days post NiV exposure. Initially, there is a high rise of temperature along with drowsiness and headache. This is followed by mental confusion as well as disorientation, ultimately progressing towards coma within 1–2 days. A critical complication of the NiV infection is encephalitis. During initial phase, the respiratory problems may become evident. There is development of atypical pneumonia. Coughing along with acute respiratory distress may be evident in certain patients (Hossain et al. 2008; Williamson and Torres-Velez 2010). There may be sore throat, vomiting, along with muscle aches (www.medicinenet.com). There may be development of septicemia along with impairment of the renal system and bleeding from the gastrointestinal tract. In severe cases within a period of 24–48 h, there may be development of encephalitis along with seizures that ultimately leads to coma (Giangaspero 2013). It is crucial to note that transmission of the virus is more common from patients having labored breathing than those having no respiratory problems (www.cdc.gov; Luby et al. 2009).
Magnetic resonance imaging (MRI) studies in human patients have revealed that the cortex, pons as well as temporal lobes of brain get involved. There may be bilateral abnormalities in the white matter of the brain. In the cerebral cortex, there may be more than one hyperintensities (T1-weighted) which are very much similar to necrosis of the laminar cortex. Lesions may also become evident in corpus callosum, brain stem, as well as cortex of the cerebrum. It is crucial to note in this regard that diffusion-weighted (DW)-MRI is employed to detect such lesions (Goh et al. 2000; Lim et al. 2002; Ang et al. 2018). There may be presence of disseminated microinfarction in the brain due to thrombosis induced by vasculitis. The neurons may also get involved directly. Vasculitic lesions of similar nature may be found in the kidneys, heart, as well as respiratory tract (Ang et al. 2018). It is also interesting to note that blood vessels of medium and small size show most involvement in case of infection due to NiV, which results in development of syncytia (multinucleated) along with fibrinoid necrosis (Ang et al. 2018).
There may be consolidation of varying degree along with hemorrhages (either petechiae or ecchymosis) in the lungs of affected pigs at necropsy. Froth-filled bronchi along with trachea are commonly observed. In certain instances, there may be presence of blood stained fluids in the trachea and bronchi. Congestion along with generalized edema is present in kidneys and brain. Both the cortex as well as suface of kidneys may become congested (Nor et al. 2000). There may be pneumonia (moderate to high) along with formation of syncytial cells in the endothelial cell lining of the blood vasculatures as revealed histologically (Chua et al. 2000; Nor et al. 2000). In the CNS and other major organs like lungs and kidneys, there may be development of small vessel vasculopathy (disseminated) in case of acute infection (Wong et al. 2009). Generalized vasculitis along with fibrinoid necrosis and mononuclear cell infiltration may be noticed in the brain, kidneys and lungs. Viral antigens at greater concentration may be present in the blood vascular endothelial cells (especially in the lungs) as is revealed immunohistologically. In the upper respiratory tract of pigs in the lumen viral antigens are evident amidst the cellular debris which is suggestive of the possible transmission of NiV through exhalation (Nor et al. 2000; Kulkarni et al. 2013). In dogs, kidneys may show congestion with severe hemorrhage. Exudates may be present in the bronchi and trachea (Nor 1999; Kulkarni et al. 2013).
Ebola virus has been raging through Western Africa for seventeen months by now. The number of new cases is declining but an end to the epidemic is not in sight. The World Health Organization registered about 27,500 cases thus far, of which 11,220 have died. It will probably take several more months until the largest EVD outbreak in history can be declared over. Guinea, Liberia, and Sierra Leone will suffer from the economic consequences for many years to come. Scientists and politicians now unanimously argue that international aid came too late and was for the most part ineffective. The WHO in particular has been facing intense criticism not having reacted appropriately to the outbreak. The horrendous images coming out of Western Africa and concern for their own safety have finally woken up the industrialized nations.
How, then, can we protect ourselves against future outbreaks of deadly microbial diseases?
Porcine reproductive and respiratory syndrome virus (PRRSV), an enveloped and positive-stranded RNA virus of Arteriviridae family, causes porcine reproductive and respiratory syndrome (PRRS). PRRS is responsible for over one billion dollar loss per year through direct and indirect costs in the US swine industry. Two entirely distinct genotypes of PRRSV circulate in European (genotype 1/PRRSV 1) and North American countries (genotype 2/PRRSV 2) and cause tremendous economic loss. PRRSV is transmitted through oral-nasal secretions and semen. The clinical signs include fever, anorexia, mild to severe respiratory problems, abortion and reproductive failures. It is the most common pathogen associated with porcine respiratory disease complex (PRDC).
Swine influenza (flu) constitutes another persistent health challenge to the global pig industry. Flu infection is caused by influenza A virus of Orthomyxoviridae family which has negative-sense, single-stranded, segmented RNA genome. Influenza virus is transmitted through direct contact with infected animals or contaminated fomites, aerosols and large droplets. The clinical signs of influenza infection include fever, anorexia, loss of weight gain and respiratory problems. Influenza associated economic losses are due to morbidity, loss of body weight gain, increased time to market, secondary infections, medication and veterinary expenses. Influenza of swine origin occasionally infect humans and can even lead to pandemics as of 2009.
Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV) are enteric pathogens of young pigs. These viruses belong to Coronaviridae family and have positive-sense, single-stranded RNA genome. TGEV did serious economic damage to the swine industry in 1990s but with the advent of vaccines it has been largely controlled. PEDV still results in high morbidity and mortality in neonatal piglets with clinical signs like severe diarrhea, vomiting, dehydration and death. In 2013/14, PEDV outbreak in the US led to over a billion-dollar loss. Rotaviruses are double-stranded RNA viruses of Reoviridae family, cause enteric infections in pigs. Rotavirus of groups A, B, C, E and H are involved in porcine enteric infections. Some of these porcine rotaviruses also have zoonotic potential.
Foot and mouth disease (FMD) is another highly contagious, acute viral disease in pigs. The etiologic agent, FMD virus (FMDV), is a positive-sense, single-stranded RNA virus of Picornaviridae family. FMDV is transmitted through direct contact with infected animals or contaminated sources. Clinical signs include high fever, appearance of vesicular lesions on the extremities, salivation, lameness and death. FMDV causes frequent epizootics in many parts of the world resulting in severe economic loss, food insecurity and trade restrictions.
Classical swine fever (CSF) or hog cholera can result in high morbidity and mortality in pigs. It is caused by CSF virus (CSFV), an enveloped, positive-sense, single-stranded virus of Flaviviridae family. Transmission of CSFV occurs through oral-nasal routes after contact with infected pigs or contaminated resources and even vertically from infected sows to piglets. Clinical signs include fever, anorexia, respiratory problems, neurological disorders, reproductive failures and death. CSF is a notifiable disease to World Organization for Animal Health (OIE). The economic losses are associated with production loss, trade limitations and tremendous expenditures in eradication programs. For example, the 1997/98 outbreak of CSFV in the Netherland resulted in death of 9 million pigs and economic losses of 2.3 billion dollars. United States is free of CSFV; however, this virus is endemic in many parts of the world including Central and South America, Africa and Asia.
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).
Over the past two decades, there has been mounting interest in the increasing number of viruses causing unexpected illness and epidemics among humans, wildlife and livestock. All too often outbreaks have seriously stretched both local and national resources at a time when health-care spending in the economically developed world has been constrained. Importantly, capacity to identify and control emerging diseases remains limited in poorer regions where many of these diseases have their origin.
Emerging disease is a term used with increasing frequency to describe the appearance of an as yet unrecognized infection, or a previously recognized infection that has expanded into a new ecological niche or geographical zone and often accompanied by a significant change in pathogenicity.1 The key message is that these are representative of constantly evolving infections responding to rapid changes in the relationship between pathogen and host.
Among 1400 pathogens of humans over 50% of these have their origins in animal species, that is, “are diseases or infections naturally transmitted between vertebrates and humans” (World Health Organization). According to Woolhouse and colleague2 emerging or re-emerging pathogens are far more likely to be zoonotic. Viruses are over-represented in this group. Moreover, viruses with RNA genomes account for a third of all emerging and re-emerging infections. Emerging pathogens are typically those with a broad host range, often spanning several mammalian orders. Almost certainly many of these infections have been the result of the development of agricultural practices and urbanization (Figure 1).
Recent interest in emerging infections has focused on three key areas. First, how the interplay of climate, environment and human societal pressures can trigger unexpected outbreaks of emerging disease. Second, the understanding of how viruses can transmit between a reservoir and new host species, Third, identifying those aspects of the disease process that offer opportunities for therapy and prevention. To these must be added a broader understanding of how viruses evolve over time, clues to which are now being uncovered through looking closely at genetic elements of the host genome responsible for resisting virus invasion. Meeting these objectives will provide a more rigorous basis for predicting virus emergence.
Influenza A, B and C viruses are members of the Orthomyxoviridae family that can cause influenza in humans. Influenza A viruses exist in humans, various other mammal species, and birds; migratory or domestic waterfowl are their largest reservoir. Humans are thought to be the primary hosts and reservoir of influenza B and C viruses, although both have been identified in other hosts after reverse zoonotic transmission from humans. While influenza B virus is a common seasonal human pathogen similar to influenza A virus in its clinical presentation, influenza C virus causes primarily upper respiratory tract infections in children. Clinical manifestations (cough, fever, and malaise) are typically mild, but infants are susceptible to serious lower respiratory tract infections. Influenza C viruses co-circulate with influenza A and B viruses and causes local epidemics,. Six genetic and antigenic lineages of influenza C viruses have been described, and as in influenza B viruses, are considered monsubtypic,. Co-circulation of multiple subtypes of influenza allows for rapid viral evolution through the process of antigenic shift, a property previously only shown for influenza A viruses. Thus, both influenza B and C viruses do not have pandemic potential. In contrast, the Influenza A genus includes 17 hemagglutinin and 9 neuraminidase subtypes, and reassortment among different subtypes has repeatedly generated pandemic viruses to which the human population is naïve–. It is the animal reservoirs of diverse influenza A viruses that give them the unique property within orthomyxoviruses of causing human pandemics.
Aside from humans, influenza C virus has been isolated only from swine in China (in 1981). Genetic analysis showed a close relation between Japanese human and Chinese swine influenza C isolates,. Serological surveys in Japan and the United Kingdom found 9.9% and 19% of swine, respectively, to have positive HI antibody titers to human influenza C viruses, suggesting that the virus is not uncommon in swine,. Swine inoculated with influenza C virus had mild respiratory disease and transmitted the virus to naive swine by direct contact. Here we characterize an orthomyxovirus isolated from a clinically ill pig and show that the virus is distantly related to human influenza C virus and readily infects and is transmissible in both ferrets and pigs. Genetic and antigenic analysis suggest that this virus represents a new subtype of influenza C virus, raising the possibility of reassortment and antigenic shift as mechanisms for influenza C virus evolution which could pose a potential threat to human health.
Clinically, influenza is not distinguishable from most other infectious diseases with fever in the tropics. In this context malaria is of particular interest when considering the African continent. In tropical Africa, malaria is an important infectious disease and is still thought to be the main cause of febrile episodes in children. However, the threshold of clinical manifestation of malaria is strongly influenced by the endemicity of Plasmodium falciparum infection in an area: in very low transmission areas, any microscopically detectable parasitemia would indicate malaria, whereas in regions of high transmission, a certain parasitemia needs to be reached to lead to malaria at least from 5 years of age on. Recent, mainly unpublished observations show that there is a considerable drop in malaria incidence and in P. falciparum prevalence rate in some African countries. Despite this reduction, the old habit of treating every child with fever with antimalarials continues. As fever due to many infectious diseases wanes after a few days without treatment, the belief that medical staff are successfully treating malaria cases lingers on.
A recent study in Lambarene, Gabon, illustrates the extent of the problem and the unresolved conundrum. In and around Lambarene all febrile children are still treated with antimalarials, mainly amodiaquine-artesunate. In a study of 1,000 consecutive children presenting with fever at our research center in Lambarene, the results from the thick blood smears indicated that only about 5% had malaria. Recent serological tests have shown that parts of the febrile illnesses in Lambarene are due not only to influenza, but also to dengue fever, chikungunya disease, and streptococcal pneumonia. In addition, in Tanzania where malaria is considered to be highly endemic, D'Acremont and coworkers refer to recent data indicating that only 10%–40% of under-5-year-old patients with fever have malarial parasites in rural areas. Thus, in Lambarene and perhaps elsewhere in Africa, the majority of febrile cases may be unnecessarily exposed to antimalarial drugs, with the well-recognized negative consequences.
It is acknowledged that the problem of diagnosing influenza-like illness is already challenging in resource-rich settings apparent from data collected on the ongoing pandemic of influenza A (H1N1, swine flu). Examining symptomatic individuals with recent history of travel to countries where the H1N1 virus was circulating indicated that other respiratory viruses such as rhinovirus, coronavirus, or para-influenzavirus were responsible for influenza-like illness. Therefore, not surprisingly, yet often ignored, there is simultaneous transmission of different respiratory viruses and bacteria in addition to malaria that lead to febrile illnesses in Africa and elsewhere in the tropics.
The task of diagnosing and treating febrile illnesses properly in resource-poor tropical settings is daunting. Yet, with attention to upgrading clinical research in Africa focused on combatting the well known diseases such as AIDS, tuberculosis, and malaria, we need to start taking a few steps towards implementing programs that deal with influenza-like illness. Thus in each of the Northern, Western, Eastern, Central, and Southern African regions one well-established research center could be identified to act as a surveillance center. Already in Senegal and South Africa such centers exist, but there is a need for identifying new ones in other regions and upgrading and intensifying activities in already existing ones. Training should ensure that epidemiological data can be gathered on attack rates, clinical spectrum of illness, and risk factors, while molecular diagnosis of collected samples confirms influenza and identifies the strain/subtypes. In close collaboration with WHO centers, the behavior of the influenza virus would then be monitored properly on the African continent. Contrary to common belief, excellent clinical research centers are developing in Africa with good epidemiologists, information technology infrastructure, and laboratory equipment.
Highly pathogenic avian influenza A (H5N1) virus infection of humans was first identified in 1997 in Hong Kong. The first case was identified in May of that year, and an additional 17 infections were detected in November and December 2017.12 Since then, H5N1 virus infections among humans have been reported in 16 countries, including 1 infection in a person who traveled to Beijing, China, and was diagnosed with pneumonia and meningoencephalitis after returning to Canada in late 2013.13 Between November 2003 and April 2017, a total of 858 infections with 453 deaths in humans attributable to highly pathogenic avian influenza A (H5N1) virus infection were reported to WHO.14 Of the 858 infections, 793 (92.4%) have been reported from 5 countries: Egypt (358 infections), Indonesia (199), Vietnam (127), Cambodia (56), and China (53).14 Of all reported infections, 52.8% (453/858) have been fatal.15 H5N1 virus infection can cause rapidly progressive pneumonia and multiple organ failure, leading to death.16 Risk factors include direct or close exposure to sick or dead infected poultry or visiting a live poultry market.17,18 Highly pathogenic avian influenza A (H5N1) virus is of major concern because case clusters representing limited, nonsustained human-to-human transmission has been reported in multiple countries,19-21 and viral evolution is ongoing in infected poultry.
Human microbiologic infections, known as zoonoses, are acquired directly from animals or via arthropods bites and are an increasing public health problem. More than two thirds of emerging human pathogens are of zoonotic origin, and of these, more than 70% originate from wildlife. In novel environments, viruses, particularly RNA viruses, can easily cross the species barrier by mutations, recombinations or reassortments of their genetic material, resulting in the capacity to infect novel hosts. Because of their adaptive abilities, RNA viruses represent more than 70% of the viruses that infect humans. When socio-economic and ecologic changes affect their environment, humans may encounter increased contact with emerging viruses that originate in wild or domestic animals.
Wolfe et al. in 2007 and Karesh et al. in 2012 described different stages in the switch from an animal-specific infectious agent into a human-specific pathogen. The key stage is the transition of a strictly animal-specific infectious agent (originating from wildlife or domestic animals) to exposed human populations, resulting in sporadic human infections (Figure 1). If the pathogen is able to adapt to its human host and acquire the means to accomplish an inter-human transmission, horizontal human-to-human transmission occurs and maintains the viral cycle. Sometimes, an intermediate host, such as a domestic animal, is the link between sylvatic viral circulation and human viral circulation. For example, some human infections originating from bats, such as Nipah, Hendra, SARS and Ebola viral infections, may involve intermediate amplification in hosts such as pigs, horses, civets and primates, respectively (Figure 1). Genetic, biologic, social, political or economic factors may explain a switch in viral host targets. For example, climate changes may influence the geographical repartition of vector arthropods, leading to new areas of the distribution of infectious diseases, like Aedes albopictus and Chikungunya infections in the Mediterranean. Morens et al. listed different key factors that may contribute to the emergence or re-emergence of infectious diseases, such as microbial adaptation to a new environment, biodiversity loss, ecosystem changes that lead to more frequent contact between wildlife and domestic animals or human populations, human demographics and behavior, economic development and land use, international travel and commerce, etc.. These patterns of transmission allow identifying different animals to follow in order to monitor the appearance of new or re-emerging infectious agents before its first detection in the human populations. Therefore, hematophagous arthropods, wildlife and domestic animals may serve as targets for zoonotic and arboviral disease surveillance, particularly because sampling procedures and long-term follow-up studies are more easily performed in these hosts than in humans.
Historically, classic viral detection techniques were based on the intracerebral inoculation of suckling mice or viral isolation in culture and the subsequent observation of cytopathic effects on cell lines. Later, immunologic methods, e.g., seroneutralization or hemaglutination, were used to detect viral antigens in various complex samples. These techniques were based on the isolation of viral agents. With the progresses of molecular biology, polymerase chain reaction (PCR)-based methods became the main techniques for virus discovery and allowed the detection of uncultivable viruses, but these techniques required prior knowledge of closely related viral genomes. Next-Generation Sequencing (NGS) techniques make it possible to sequence all viral genomes in a given sample without previous knowledge about their nature. These techniques, known as viral metagenomics, have allowed the discovery of completely new viral species. Because of their low cost, the use of NGS techniques is exponentially increasing.
The transmission of infections between humans occurs after a pathogen from a wild or domestic animal contacts with exposed human populations. The human exposures may or may not be mediated by the bite of bloodsucking arthropods. Surveillance programs may target wildlife, domestic animals or arthropods for emerging viruses before their adaptation to human hosts.
African swine fever (ASF) is a severe viral disease that manifests clinical symptoms of hemorrhagic fever caused by African swine fever virus (ASFV) and can result in case fatality rates of up to 100% in domestic pigs, depending on the virus strain (Galindo and Alonso, 2017). ASF, which was first identified by Montgomery (1921) in Kenya in the 1920s, made its first incursion into Europe via two successive entries into Portugal (in 1957 and again in 1960), spreading rapidly throughout Western Europe and then to South America and the Caribbean (Michaud et al., 2013). It was eventually eradicated by the mid-1990s, with the exception of Sardinia (Mur et al., 2016). However, since the second major incursion of the disease, initially into Georgia in 2007, ASF has spread to Eastern Europe and Russia (Rowlands et al., 2008; Revilla et al., 2018). The virus has continued to spread worldwide, including China. Since the first ASF case emerged in China in August 2018, more than 100 cases have been recorded in 25 provinces (Zhou et al., 2018).
African swine fever normally presents with non-specific symptoms, including fever, anorexia, vomiting, and diffused hemorrhage in superficial skin. Post-mortem examination shows pericardial effusion, kidney enlargement, lymphadenectasis, and darkened and enlarged spleen. All these features are indistinguishable from those seen in classical swine fever virus (CSFV) infection (Tauscher et al., 2015; Li and Tian, 2018). At present, no effective treatment or vaccine for ASFV is available, and disease control is based mainly on animal slaughtering and strict sanitary measures (Cisek et al., 2016; Rock, 2017). Rapid laboratory diagnosis is important for timely triage and confirmation to control and preventing this disease because of its rapid progression to death and spread.
Molecular tools based on the detection of the genetic information of ASFV have become more widely accepted for ASF diagnosis (Oura et al., 2013). Polymerase chain reaction (PCR) and real-time PCR techniques have provided a supportive method to post-mortem ASF diagnosis (Aguero et al., 2003; Zsak et al., 2005; Fernandez-Pinero et al., 2013); however, they cannot be used for field (on-site) detection in pig farms. Recently, Liu et al. (2019) reported that the improved real-time PCR assay using a Universal Probe Library (UPL) probe could be applied to ASFV molecular diagnosis under field conditions. Due to limitations of the battery-powered real-time PCR instrument, it can process only a moderate number of samples. Isothermal recombinase polymerase amplification (RPA) has been successfully used to detect multiple viral pathogens, including infectious bovine rhinotracheitis virus (Hou et al., 2017), bovine coronavirus (Amer et al., 2013), Ebola virus (Yang et al., 2016), bovine viral diarrhea virus, or foot-and-mouth disease virus (Wang et al., 2018). As reports of ASFV detection by RPA are limited, we aimed to develop and evaluate a rapid detection tool that combines immunochromatographic strip tests, more commonly referred to as lateral flow devices (LFDs), with RPA targeting the conserved ASFV p72 gene.
Some of the most widely studied epidemiologic models are for influenza, both pandemic and seasonal. Because of the fleeting herd immunity characteristic of flu, such models typically use a deterministic compartmental disease model, in which individuals move from susceptible, to infectious, to recovered, and once again become susceptible (SIRS). For seasonal flu, which appears in the northern hemisphere every winter and virtually disappears during the summer months, the subtlety involved in modeling relates to the way seasonality is introduced, and how that seasonality may vary for different strains of the virus.9-11 Predictions of upcoming flu seasons are critical to vaccine development but remain imprecise.
Working with Israeli researchers, members of the STEM community developed and ran 3 models using 10 years of incidence data for seasonal flu collected by the Israeli Center for Disease Control from health clinics across the country.12,13 In 8 years, strain A was more frequently identified; in 2 years, strain B. The first model (a control) assumed no differences between the 2 strains; the second allowed for different transmission rates; and the third expanded to include different background transmission and flu season length. Using STEM's automated experiment (Nelder-Mead) plug-in,14 the project team cross-validated the models by fitting them to the historic data for a subset of the 10 years and then attempting to predict the incidence for the remaining years. Results showed the second model, which accounted for variations in transmission, increased the predictive ability for both strains, while the third model did not.
The team concluded that the next step to improve predictive capabilities would be to fit a full multistrain model of influenza, but they questioned whether the available data were adequate to do so.13
Insect-borne diseases are infectious diseases spread among animals and human through hemophagia by blood-feeding arthropods, such as mosquitoes, ticks and midges, which are widespread in the environment. These infections lead to significant human and animal morbidity and mortality worldwide, which cause a huge economic loss. In recent years, epidemic outbreaks of insect-borne infectious diseases in countries neighboring and trading with China have posed a threat to public health, especially in the border areas of China. Many insect-borne diseases in the border areas of China have drawn public attention, such as West Nile virus (WNV) infection in Xinjiang, Tahyna virus infection in the Qinghai-Tibet Plateau, and mosquito-borne arbovirus (e.g. Japanese encephalitis virus and Sindbis virus) infection in Yunnan. It is remains difficult to develop effective vaccines against such viruses. Furthermore, the clinical symptoms of many infections by mosquito-borne arboviruses do not indicate a specific pathogen, and some infections are even asymptomatic. Therefore, accurate and timely diagnosis of these infections is a great challenge of utmost importance.
To date, multiple molecular detection methods have been established to detect insect-borne pathogens, including reverse transcriptase-polymerase chain reaction (PCR), real-time PCR, a liquid bead array and a microwell membrane array. Recent studies have established modified PCR or array methods for the detection of insect-borne pathogens [11–13]. However, these methods are only able to detect one type or a few types of viruses, which greatly restricts their application. Luminex xMAP technology is a multiplexed high-throughput detection system that uses fluorescent carboxylated microspheres. The Luminex array offers up to 100 independent channels and use microspheres embedded with various ratios of two fluorescent dyes. A mixed suspension of microspheres is mixed with the sample to bind analytes, which are then labeled with a fluorescent reporter and analyzed using a specialized flow cytometer [14, 15].
In this study, we established a method that was able to simultaneously detect multiple insect-borne pathogens rapidly and effectively, including bluetongue virus (BTV), epizootic hemorrhagic disease virus of deer (EHDV), Q-fever pathogen Coxiella burnetii (CB), African swine fever virus (ASFV), West Nile fever virus (WNV), Borrelia burgdorferi (BB), vesicular stomatitis virus (VSV), Rift Valley fever virus (RVFV), Ebola virus (EBV) and Schmallenberg virus (SBV). This optimized liquid array detection system may contribute to the rapid and effective detection of multiple insect-borne diseases at border ports in China.
Rotaviruses (RVs) were first isolated in 1973 from children in Australia. After the identification in swine two years later, RVs were recognized as the major etiological agents of acute viral gastroenteritis in humans and domesticated livestock worldwide. Belonging to the Reoviridae family, the RV genome is composed of 11 double stranded RNA segments. Eight RV species (RVA-RVH) and two tentative species (RVI and RVJ) have been identified by sequence-based classification of inner capsid protein 6 (VP6). RVA, RVB, RVC and RVH have been detected in both humans and animals while RVD-RVG, RVI, and RVJ have only been found in animals. Five out of ten RV species have been described in pigs (RVA, RVB, RVC, RVE, and RVH).
Of the RV species, RVA is most common and well characterized both in animals and humans due to its high prevalence and pathogenicity. Porcine RVA was isolated in 1975 followed by identification of swine RVC and RVB. A recent two-year study found RVB in 31.8% of diarrheic samples from North American swine, indicating higher detection of RVB than previously observed. Similar detection rates of porcine RVB (25.9%) have been identified in Japan. Although identified at lower rates than in North America and Japan, swine RVB has also been detected in Europe, South Africa, India, and Brazil.
Despite unexpectedly high detection rates of RVB in swine, RVB pathogenesis has only been established in gnotobiotic and caesarian-derived colostrum-deprived piglets. The inability to cultivate RVB and limited whole genome sequence data has hampered an understanding of transmission and evolution within pigs. In order to fill these knowledge gaps, this study used metagenomic sequencing to identify porcine RVB from an enteric outbreak in a farm from southern Siberia, determined its disease-causing ability using experimental inoculation experiments, and studied its phylogenetic relationship with previously characterized swine RVB strains.
Porcine epidemic diarrhea (PED) is an acute, highly contagious and devastating enteric disease characterized by severe enteritis, vomiting and watery diarrhea in swine. PED is caused by porcine epidemic diarrhea virus (PEDV), which was firstly identified in Belgium in 1978. PEDV is an enveloped, single-strand, and positive-sense RNA virus, which belongs to the Coronaviridae family. The PEDV genome is ~28 kb in length and comprised of a 5′ untranslated region (UTR), a 3′ UTR, and at least seven open reading frames (ORFs) that encode four structural proteins [spike (S),envelope (E), membrane (M),and nucleocapsid (N)] and three non-structural proteins(replicase 1a and 1b, and ORF3).
In China, PED was firstly occurred in Shanghai in 1973. So far, PEDV has been observed on most swine breeding farms in most provinces since late 2010 in China.The economic losses caused by PEDV infection have been continuous and serious in China. Recently, PEDV has suddenly emerged in the United States and rapidly spread across the country, resulting in high mortality in infected newborn piglets, which have posed serious economic losses to the swine industry in the USA [7, 8]. Rapid diagnosis and timely monitoring of potential PED outbreaks are among the first important steps in the prevention and control of PED. Currently, several conventional methods are available for the detection of PEDV, including virus isolation, fluorescence assay, immune electron microscopy, enzyme-linked immuno- sorbent assay and molecular biological characterization. However, the isolation and identification of viruses require extended periods of time ranging from days to weeks; so this method does not meet the time requirements needed for the prevention of epidemics. Therefore, these rapid, sensitive and specific molecular biological techniques, including RT-PCR and real-time RT-PCR, have played important roles in the rapid detection of PEDV. Nevertheless, all of these techniques require sophisticated instrumentation (such as PCR machines and quantitative fluorescence PCR machines), limiting the effectiveness of these procedures in smaller, under-equipped laboratories.
The loop-mediated isothermal amplification (LAMP) technique is a molecular biology method used to amplify specific DNA fragments in vitro. This method only requires a water bath or heating block to amplify large amounts of nucleic acids in 30 ~ 60 minutes without additional expensive equipments. In addition, there is no need to use nucleic acid electrophoresis to assess the result, for the reason that the result can be easily observed in the presence of a fluorescent dye. These characteristics make the LAMP method a simple, fast, effective and practical DNA amplification method, which has been successfully implemented for the detection of avian influenza A viruses, porcine reproductive and respiratory syndrome virus, foot-and mouth disease virus and PEDV. The PEDV M protein, the most abundant envelope component, is a triple-spanning membrane glycoprotein with a short amino-terminal domain outside of the virus and a long carboxy-terminal domain inside. The M protein plays an important role in the virus-assembly process, and induces antibodies that neutralize the virus in the presence of its complement. In this study, five primer sets were designed based on the conserved regions of the M gene, and a real-time RT-LAMP method was developed for the detection of PEDV.
Coronaviruses are large enveloped viruses with a single-stranded positive-sense RNA genome. They can infect humans, as well as a variety of animals, such as bats, mice, birds, dogs, pigs, and cattle, causing mainly respiratory and enteric diseases. The virus MERS-CoV is a new member of the beta group of coronavirus, Beta coronavirus. MERS-CoV is different from SARS coronavirus and different from the common-cold coronavirus and known as endemic human betacoronaviruses HCoV-OC43 and HCoV-HKU1. MERS-CoV had frequently been referred to as a SARS-like virus, or the novel coronavirus until 23 May 2013. On September 11, 2012, a 49-year-old man from Qatar, with a history of travel to Saudi Arabia, was transferred to the United Kingdom with symptoms of severe respiratory illness. Sample from the lower respiratory tract samples of the patient was found positive after a pan-coronavirus RT-PCR assay. Comparison of the sequence of the PCR fragments with the ones obtained in the case of the Saudi patient revealed that they share 99% similarity, suggesting infection by the same virus. Sequencing of the novel coronavirus was performed at the Erasmus Medical Center (EMC) in Rotterdam, the Netherlands, where the virus was named “human coronavirus EMC” (hCoV-EMC). Later, the Coronavirus Study Group of the International Committee on Taxonomy of Viruses renamed the virus “Middle East respiratory syndrome coronavirus” (MERS-CoV) in May 2013.
MERS-CoV is a zoonotic virus that is transmitted from animals that are a reservoir of the virus to humans. Although the source of MERS-COV is not elucidated yet, camels are the most likely source of infection in human [3, 4]. A coronavirus similar to large extent to the one detected in humans has been isolated from camels in Egypt, Oman, Qatar, and Saudi Arabia. Other animals including goats, cows, sheep, water buffalo, swine, and wild birds, have been tested for MERS-CoV and only dromedary camels have evidence of sero-positivity to MERS-COV, supporting the premise that dromedary camels are a likely source of infection in humans.
Direct contact with the saliva of infected camels, or consumption of contaminated milk or meat was the suspected transmission route for human MERS-COV infection. Previous study found that the seroprevalence of MERS-CoV was several times higher in persons with regular exposure to camels than in the general population [5, 6]. However, there are some cases infection in which the patients do not have any contact with sick animals or their products. This might be human to human route of transmission.
Only limited numbers of zoonotic diseases have been reported to be transmitted from human to human. The incapability of MERS-CoV to infect animal models like hamsters, mice, and ferrets, indicates the presence of a species barrier. However, an experimental study showed that human cell lines were susceptible to MERS-CoV infection, and the reports of human-to-human transmission have increased [9–11]. The modes of human-to-human MERS-CoV spread are incompletely defined.
Emergence of the Middle East respiratory syndrome coronavirus (MERS-CoV) has caused significant concern. A total of 635 laboratory-confirmed cases of MERS-CoV infection have been reported globally, including 193 deaths. Cases of MERS-CoV in Saudi Arabia were reported for the first time in September 2012, following the death of a patient due to a severe respiratory illness [13–15]. Little is known regarding the extent of human infection or the degree of detection bias towards more severe cases. If the severe cases currently being detected represent only a small sentinel minority of a much larger population of milder cases (as occurred in the early stage of the 2009 H1N1 pandemic in Mexico), the case-fatality ratio might be substantially lower than what current surveillance data suggest. Conversely, for the severe acute respiratory syndrome (SARS) epidemic of 2003, there was little evidence of the existence of undetected mild or subclinical infections [17–20].
Risk factors for the disease in humans are incompletely understood, although MERS is proposed to be a zoonotic disease. Dromedary camels have been implicated due to reports that some confirmed cases were exposed to camels. In the Middle East, confirmed cases of MERS-CoV have arisen as sporadic, familial, or hospital clusters [13, 22–26]. Although human-to-human transmission of MERS-CoV has been identified in many European, African, and Middle Eastern countries, [27–30] a genomic analysis of the Riyadh MERS-CoV isolates suggested that there were three genetically distinct lineages of MERS-CoV; therefore, it was unlikely that the Riyadh infections were the result of a single, continuous chain of human-to-human transmission [15, 31, 32]. A recent study provided further evidence of non-sustainable transmission among humans and suggested that transmission within Saudi Arabia was dependent on contact with an animal reservoir or animal products; however, no animal reservoir has yet been identified. While contact with camels has been reported, these reports were limited to only the primary cases [22, 33–37]. The MERS-CoV was related with a strain of severe acute respiratory syndrome coronavirus (SARS-CoV) that caused an outbreak in 29 countries in 2002–2003. This outbreak was characterized by 8273 cases and 775 deaths, with the majority of cases in Hong Kong. As was determined for the SARS-CoV during its pre-pandemic stage, the MERS-CoV has likely been repeatedly transmitted from an unknown animal host to humans in the past year [17–20].
To obtain effective control of the MERS-CoV outbreak, the MOH of the Kingdom of Saudi summoned a Rapid Response Team (RRT). The RRT was composed of 15 infectious disease doctors and two infection control professionals affiliated with the Korean Society for Infectious Diseases and the Korean Society for Healthcare-associated Infection Control and Prevention. The RRT established national infection control and prevention guidelines for the diagnosis and management of MERS-CoV infections [32, 38–41]. The current study aimed to investigate the demographic characteristics, history of contact with camels or positive cases, mortality, clinical manifestation and comorbidities for confirmed cases of MERS-CoV.
Although acute respiratory illness is a major cause of morbidity and mortality among children in sub-Saharan Africa, it has received relatively little attention. This is unfortunate, as underlying diseases such as AIDS, malaria and tuberculosis, which are highly prevalent in the region, can worsen such illnesses. The respiratory viruses known to cause acute illness include human respiratory syncytial virus (HRSV), human parainfluenza virus (PIV), human metapneumovirus and influenza viruses. Until recently, the burden of influenza and influenza-like illness in Africa was considered to be negligible, mainly because of the lack of confirmation assays. Reports from Cameroon and Senegal, however, show that influenza viruses are actively circulating and may be causing regular epidemics.
A clear picture of the contribution of each pathogen to acute respiratory illness is needed in order to improve prevention and clinical management and consequently to reduce the burden of disease. The emergence of the novel influenza A/H1N1 of swine origin in Mexico in April 2009 and its rapid spread worldwide, causing a global pandemic, led the health authorities of the Central African Republic (CAR) to collaborate with the World Health Organization in strengthening biological surveillance of acute respiratory illness.
The aim of the study reported here was to determine the circulation of 2009 pandemic influenza A/H1N1 virus (H1N1pdm09) by molecular methods and to identify the causative viruses, the incidence and the clinical features of acute respiratory illness among infants and young children at sentinel sites in Bangui and three rural areas.
All infants and children aged between 0–15 years who attended sentinel sites in Bangui and three rural areas (Figure 1) for influenza-like illness (ILI) or severe acute respiratory illness between January and December 2010 were included in the study (Figure 2A). The World Health Organization definitions were used for ILI (sudden onset of fever of > 38°C and cough or sore throat in the absence of other diagnoses) and severe acute respiratory illness (ILI symptoms and shortness of breath or difficulty in breathing and requiring hospital admission). The study protocol was approved by the National Ethics Committee of the CAR. Individual written informed consent was sought from the parents or guardians of all participants.
Nasopharyngeal samples were collected from 329 infants and children and within 48 hrs at 4°C to the National Influenza Centre by using rayon-budded swabs with virus transport medium pre-impregnated sponge (Virocult, Medical Wire & Equipment, UK).
RNA was extracted with a QIAmp RNA mini kit (Qiagen) according to the manufacturer’s instructions. Influenza A viruses were detected with a previously described assay targeting the conserved matrix gene for universal detection of these viruses, and H1N1pdm09 virus was identified with a specific one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) assay (designed by the National Influenza Centre of northern France, Institut Pasteur, Paris; primers and probe available upon request at email@example.com). All specimens were also tested for other respiratory viruses in two previously described multiplex semi-nested RT-PCR assays for detecting influenza A and B viruses, HRSV, human metapneumovirus and PIV types 1, 2, 3 and 4. All assays were performed on an ABI 7500 platform (Applied Biosystems, Foster City, California, USA) with the SuperScript III Platinum One-step Quantitative RT-PCR System (Invitrogen, Carlsbad, California, USA). A specimen was considered positive if the signal curve crossed the threshold line within 40 cycles. The assay limit of detection for pandemic H1N1pdm09 influenza virus is of order of magnitude of 10 copies/μL of initial sample. The assay limit of detection for influenza A and B viruses, HRSV, human metapneumovirus and PIV-3 is of order of magnitude of 10 copies/μL. For PIV-4, the assay limit of detection is of order of magnitude of 100 copies copies/μL, and for PIV-1 and −2, 1000 copies/μL. After amplification, the PCR products were purified and sent to GATC Biotech (Konstanz, Germany) for sequencing.
Student’s t test and the Pearson chi-squared test were used to assess intergroup differences. Statistical analyses were performed with EpiInfo software (V 3.5.1 CDC). A test was considered significant when the p value was < 0.05. The newly obtained sequences were analysed and compared with sequences available in GenBank.
A confirmed case was defined as any person with laboratory confirmation of MERS-CoV infection based on positive real-time polymerase chain reaction (PCR) of MERS-CoV in swab samples collected by the MOH in addition to any one of the following clinical definitions: fever (>38 °C), a cough, shortness of breath (SOB), sore throat, vomiting, diarrhea, hemoptysis, chest pain and/or infection, respiratory failure, loss of consciousness, runny nose and any asymptomatic outpatients with a history of contact with positive symptomatic cases and tested positive. Patients provided their signed consent to publication where legal guardian provided consent for a minor.
Some of the worst haemorrhagic fever outbreaks in humans, great apes and nonhuman primates (NHPs) have been caused by Ebola viruses (EBOV) and Marburg virus (MARV). These enveloped filamentous, negative sense single-stranded RNA viruses belong to the family Filoviridae. The African EBOV species, including Zaire ebolavirus (ZEBOV), Sudan ebolavirus (SEBOV), Cote d’Ivoire ebolavirus (CIEBOV) and Bundibugyo ebolavirus (BEBOV) have been associated with disease of varying morbidity and mortality in humans. So far ZEBOV has had the highest case fatality rate ranging from 60 to 90%. The only EBOV species found outside of Africa is Reston ebolavirus (REBOV). Though REBOV can infect humans, it has never been associated with clinical disease in people. However, it can cause disease and mortality in cynomolgus macaques.
Until recently, NHPs were considered the only animal species susceptible to REBOV. In 2008, REBOV was detected in pigs co-infected with porcine respiratory and reproductive syndrome virus (PRRSV) in the Philippines. Antibodies to REBOV were also detected in humans that had been in close contact with the infected pigs, suggesting pig to human transmission. Since these pigs were co-infected with PRRSV, it was not clear whether the observed clinical signs were due to REBOV or the co infecting virus. Subsequent experimental challenge suggested that REBOV infection in pigs may be asymptomatic despite replication of the virus in the lungs and shedding in nasopharyngeal secretions. Nevertheless, the emergence of REBOV in pigs had prompted our group to investigate whether the African EBOV species could also infect pigs. Using experimental challenge with the highly pathogenic ZEBOV, we observed clinical disease in pigs in the form of fever, severe respiratory distress characterized by rapid abdominal breathing; inappetence and lethargy. In addition, the infected pigs could transmit virus to contact animals. Gross pathological lesions included pulmonary consolidation, enlarged and sometimes haemorrhagic lung associated lymph nodes. Histopathology lesions consisted of alveolitis characterized by haemorrhage, presence of fibrin, neutrophils and macrophages in alveoli as well as inflammatory cells in thickened alveolar walls. Viral antigen was detected by immunohistochemistry in bronchiolar and alveolar epithelial cells, endothelial cells, macrophages and in inflammatory exudates in trachea. Apparently, experimental ZEBOV and REBOV in pigs seem to target the respiratory system but only the former induces overt clinical disease,.
In humans and non-human primates (NHPs), ZEBOV causes a severe systemic disease and dysregulated serum proinflammatory cytokine response is often linked to the disease pathogenesis. Changes in peripheral leukocytes subsets have also been observed in human and NHP ZEBOV infections.
The first study indicated local cytokine responses and cellular changes in the lungs of ZEBOV-infected pigs. Proinflammatory cytokines (IFN-γ, IL-6, IL-8 and TNF-α) were induced early while INF-α was significantly down-regulated in these lungs. Cellular changes included the infiltration of neutrophils, macrophages and lymphocytes into alveoli, with macrophages being the only leukocyte subset in the lungs that stained positive for ZEBOV. The aim of the current report was to further elucidate the mechanisms involved in the cellular infiltration into the lungs, by using a porcine microarray to analyze changes in mRNA transcription for a range of proinflammatory cytokines, chemokines and acute phase proteins in porcine lungs. In addition, we aimed to characterize the cellular infiltrates into the lungs using cell type-specific surface markers, also permitting the definitive identification of the cells harbouring ZEBOV antigen. Furthermore, we attempted to evaluated changes in PBMC subsets and serum cytokines.