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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).
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.
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).
Pigs also suffered during the 1998/99 Malaysian outbreak, but this was only diagnosed as part of the investigation following the human cases. The severity of symptoms of NiV infection in pigs varied with age. In suckling pigs (<4 weeks old), mortality could be high (up to 40%) and labored breathing and muscle tremors were evident. In growing pigs (1 to 6 months), an acute febrile (>39.9°C) illness was observed with respiratory signs ranging from increased or forced respiration to a harsh, loud non-productive cough, open mouth breathing, and epistaxis (26). In some cases these respiratory signs were accompanied by one or more of the following neurological signs: trembles, neuralgic twitches, muscle fasciculation, tetanic spasms, incoordination, rear leg weakness, or partial paralysis. Pigs of this age had high morbidity and low mortality (<5%) (26–28). Some animals over 6 months of age died rapidly (within 24 h) without signs of clinical disease. Respiratory signs were reported in adult pigs, as with younger animals, although these were less obvious (labored breathing, bloody nasal discharge, increased salivation) and neurological signs included head pressing, bar biting, tetanic spasms and convulsions. First trimester abortions were also reported (26–28).
In an experimental infection study, pigs were inoculated subcutaneously with a NiV isolate from the central nervous system of a fatally infected human patient. Infection elicited respiratory and neurological symptoms consistent with those observed in naturally infected Malaysian pigs, which included febrile illness, incoordination, mucosal nasal discharge, and persistent cough (29). Pigs inoculated orally with the same dose did not show clinical signs although they still shed virus. In a second study, piglets were inoculated oronasally with a human NiV isolate (30). All infected animals showed a transient increase in body temperature between 4 and 12 days post-infection. Two of these animals developed transient respiratory signs, mild depression and a hunched stance. Both these studies concluded that NiV infection in pigs had no pathognomonic features i.e., the clinical signs observed were non-specific. This can make field diagnosis of NiV infection in pigs difficult, as observed in the outbreak in Malaysia (16, 28).
The name proposed for the disease caused by NiV infection of pigs was “porcine respiratory and neurological syndrome” (also known as “porcine respiratory and encephalitis syndrome”), or, in peninsular Malaysia, “barking pig syndrome” (28). NiV infection was included as the sixth pig disease notifiable to the OIE World Organization for Animal Health (31). The OIE approve diagnostics and recommends preventative and control measures for a range of transboundary livestock diseases.
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.
The porcine hemagglutinating encephalomyelitis virus (PHEV) is the causative agent of neurological and/or digestive disease in pigs. PHEV was one of the first swine coronaviruses identified and isolated, and the only known neurotropic virus that affects pigs. However, PHEV remains among the least studied of the swine coronaviruses because of its low clinical prevalence reported in the swine industry worldwide. PHEV can infect naïve pigs of any age, but clinical disease is age-dependent. Clinical manifestations, including vomiting and wasting and/or neurological signs, are age-related, and generally reported only in piglets under 4 weeks old. Subclinical circulation of PHEV has been reported nearly worldwide in association with a high seroprevalence in swine herds. Protection from the disease could be provided through lactogenic immunity transferred from PHEV seropositive sows to their offspring in enzootically infected herds. However, PHEV still constitutes a potential threat to herds of high-health gilts, as evidenced by different outbreaks of vomiting and wasting syndrome and encephalomyelitis reported in neonatal pigs born from naïve sows, with mortality rates reaching 100%. In absence of effective vaccine, the best practice for preventing clinical disease in suckling piglets could be ensuring that gilts and sows are PHEV seropositive prior to farrowing.
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
Nipah virus (NiV) is an enveloped, single stranded, negative sense RNA paramyxovirus, genus Henipavirus. The natural hosts and wildlife reservoirs of NiV are Old World fruit bats of the genus Pteropus (1). Both Nipah and the related Hendra virus possess a number of features that distinguish them from other paramyxoviruses. Of particular note is their broad host range which is facilitated by the use of the evolutionary conserved ephrin-B2 and –B3 as cellular receptors (2). The NiV attachment glycoprotein (G) is responsible for binding to ephrin-B2/-B3 (3). Following receptor binding, the G protein dissociates from the fusion (F) protein. Subsequently, the F protein undergoes a series of conformational changes which in turn initiates fusion of the viral and host membrane allowing entry (4). During viral replication, the F protein is synthesized and cleaved into fusion active F1 and F2 subunits. These subunits are subsequently transported back to the cell surface to be incorporated into budding virions, or facilitate fusion between infected and adjacent uninfected cells (5). This cell-to-cell fusion results in the formation of multinucleated cells called syncytia, and greatly influences the cyopathogenicity of NiV as it allows spread of the virus, even in the absence of viral budding (5, 6).
NiV infection is currently classed as a stage III zoonotic disease, meaning it can spill over to humans and cause limited outbreaks of person-to-person transmission (7, 8). NiV outbreaks have been recognized yearly in Bangladesh since 2001 as well as occasional outbreaks in neighboring India (Figure 1). These outbreaks have been characterized by person-to-person transmission and the death of over 70% of infected people (10, 11). In May 2018, the first ever outbreak in southern India was reported. A total of 19 NiV cases, of which 17 resulted in death, were reported in the state of Kerala. Pteropus giganteus bats from areas around the index case in Kozhikode, Kerala, were tested at the National High Security Animal Diseases Laboratory at Bhopal. Of these, 19% were found to be NiV positive by RT-PCR (12). Characteristics of NiV that increase the risk of it becoming a global pandemic include: humans are already susceptible; many NiV strains are capable of person-to-person transmission; and as an RNA virus, NiV has a high mutation rate (13). NiV has been found to survive for up to 4 days when subjected to various environmental conditions, including fruit bat urine and mango flesh (14). Whilst survival time was influenced by fluctuations in both temperature and pH, the ability for NiV to be spread by fomites could play a role in outbreak situations.
The first and still most devastating NiV outbreak occurred in peninsular Malaysia from September 1998 to May 1999 (15, 16). The link to pigs in this outbreak was obvious as 93% of the infected patients had contact with pigs (17). If a NiV strain were to become human-adapted and infect communities in Southeast Asia where there are high human and pig densities and pigs are a primary export commodity, infection could rapidly spread and humanity could face its most devastating pandemic (8, 11, 18).
Viral diseases affect millions of people worldwide. Annually, dengue virus disease affects about 50 to 100 million people globally with 9000+ fatalities, rotavirus infects about two million children under five years of age, of whom about 527,000 die, seasonal influenza epidemics cause severe illness in three to five million people, and a quarter to a half million deaths, just to name a few. While drugs and vaccines are available for many of the viral diseases, the high mutation rate characteristic of viral genomes renders many of these rapidly obsolete. There is thus a continuous hunt for new drugs and vaccines, and this is compounded by the fact that new viruses are coming up to attack human hosts with higher frequency, while mutability of viral sequences rapidly render existing drugs and vaccines obsolete. Among the latest incidents of viral epidemics, one may recall the H1N1 (Influenza A type with Hemagglutinin subtype 1 and Neuraminidase subtype 1) swine flu pandemic of 2009, the SARS (Severe Acute Respiratory Syndrome) epidemic of 2002–2003, the MERS (Middle East Respiratory Syndrome) epidemic of 2015, the Ebola epidemic of 2014–2015 with 28,639 cases and 11,316 deaths reported until 16 March 2016, the dengue epidemics in India of 2015, and now, in 2016, the Zika virus epidemic in South America.
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.
PHEV can infect naïve pigs of any age, but clinical disease is variable and dependent on age, possible differences in virus virulence (74), and the course of viral pathogenesis. In growing pigs and adults, PHEV infection is subclinical, and animals develop a robust humoral immune response against the virus (66, 75). Exceptionally, transient anorexia (1–2 days) was reported in PHEV-infected sows in absence of other clinical signs (55). An experimental study performed on 7 weeks old pigs reported transient mild neuromotor signs, including tremor and generalized muscle fasciculation in 17% (2/12) of pigs between 4 and 6 days after oronasal inoculation (75). Acute outbreaks of VWD and encephalomyelitis have been reported in piglets under 3–4 weeks of age born from naïve sows, with mortality rates reaching 100%. The first signs of infection are generally non-specific and may include sneezing and/or coughing because of virus replication primarily occur in the upper respiratory tract; followed by transient fever that may last for 1–2 days. More specific clinical manifestations may appear between 4 and 7 days after infection and are characterized by (1) VWD and (2) neurological signs including tremor, recumbency, padding opisthotonus, and finally death. Both clinical forms can be observed concurrently in the same herd during an acute outbreak. More recently, PHEV was associated with a case of influenza-like respiratory illness in a swine exhibition in Michigan, USA, in 2015 (76). Although PEHV can replicate in the respiratory epithelium, the role of PHEV as respiratory pathogen has not yet been confirmed and needs further investigation.
The VWD was experimentally reproduced and reported for the first time in 1974 (59) in colostrum-deprived (CD) pigs by oronasal and intracranial inoculation. Mengeling et al. (74) experimentally reproduced both clinical forms of the disease in neonatal pigs inoculated with a field virus isolate. Later, Andries et al. (77) evaluated the clinical and pathogenic outcomes with different routes of inoculation. In this experiment, all piglets inoculated oronasally or via the infraorbital nerve showed signs of VWD 5 days after the inoculation. However, a high percentage of animals inoculated through the stomach wall, intramuscularly, and intracerebrally showed VWD signs 3 days after inoculation. Pigs inoculated intravenously, intraperitoneal or in the stomach lumen did not show PHEV-associated VDW signs.
Suckling piglets experiencing PHEV-associated VWD show repeated retching and vomiting, which could be centrally induced (4, 49, 59, 73). The persistent vomiting and decreased food intake result in dehydration, constipation, and therefore a rapid loss of body condition. PHEV-infected neonates become severely dehydrated after few days, exhibit dyspnea, cyanosis, lapse into a coma, and die. During the acute stage of VWD outbreaks, some pigs may also display neurologic signs, including muscle tremors, hyperesthesia, excess physical sensitivity, incoordination, paddling, paralysis, and dullness (68). When the infection occurs in older pigs, there is anorexia followed by emaciation (Figure 1). They continue to vomit, although less frequently than in the acute stage. After the acute stage, animals start showing emaciation (“wasting disease”) and often present distension of the cranial abdomen. This “wasting” state may persist for several weeks after weaning, which in most cases requires euthanasia.
Pre-weaning morbidity varies depending on the immune status of neonatal litters at the time of PHEV infection (4, 74). In piglets without lactogenic immunity against PHEV, morbidity is litter-dependent and may approach 100% when the infection occurs near birth. Overall, morbidity decreases markedly as the pig's age increases at the time of PHEV infection. Mortality is variable, reaching up to 100% in neonatal litters born from PHEV naïve dams. However, a different epidemiological picture was observed in the outbreak reported during the winter of 2006 in Argentina (66) where only suckling pigs born from an isolated pool of non-immune gilts were affected. The severity of the main clinical signs reported, including vomiting, emaciation, wasting, and death was unexpected according to previous reports in the field (73). The morbidity was 27.6% in 1 week old pigs and declined to 1.6% in 3 weeks old pigs. After weaning, 15–40% of the pigs coming from affected farrowing units showed wasting disease. An estimated 12.6% (3,683) pigs died or were euthanized (66).
The first clinical signs observed during neurological PHEV outbreaks include sneezing, coughing, and vomiting 4–7 days after birth, with a morbidity rate of approximately 100% (4, 78, 79). Mild vomiting may continue intermittently for 1–2 days. In some outbreaks, the first sign is acute depression and huddling. After 1–3 days, pigs exhibit various combinations of neurological disorders. Generalized muscle tremors and hyperesthesia are common. Pigs may have a jerky gait and walk backwards, ending in a dog-sitting position. They become weak and unable to rise, and they paddle their limbs. Blindness, opisthotonus, and nystagmus may also occur. Finally, the animals become dyspneic and lie in lateral recumbency. In most cases, coma precedes death, with a mortality rate of 100% in neonatal pigs (4). Older pigs show mild transient neurological signs, including generalized muscle fasciculation and posterior paralysis. Outbreaks described in Taiwan (65) in 30–50 days old pigs were characterized by fever, constipation, hyperesthesia, muscular tremor, progressive anterior paresis, posterior paresis, prostration, recumbency, and paddling movements with a morbidity of 4% and a mortality of 100% at 4–5 days after the onset of clinical signs.
In non-swine species, PHEV-related disease only has been induced experimentally. It was also demonstrated that suckling mice (3 days old) were susceptible in a dose- and age-dependent manner to PHEV infection through intracranial inoculation, showing neurological signs and dying (80).
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.
The caliciviruses (family Caliciviridae) are non-enveloped, positive sense, single-stranded RNA viruses with diameters ranging from 27 to 40 nm. Caliciviruses cause a wide range of significant diseases in human and animals. At present, there are five recognized genera, i.e., Norovirus, Sapovirus, Lagovirus, Vesivirus, and Nebovirus with several additional candidate genera or species proposed and under evaluation by the International Committee on Taxonomy of Viruses (ICTV) [1, 2] (http://www.caliciviridae.com/unclassified/unclassified.htm). In the Vesivirus genus, Vesicular exanthema of swine virus (VESV) and Feline calicivirus (FCV) are two species currently approved by ICTV. Several canine caliciviruses (CaCV) isolates have been identified and shown to be phylogenetically related to vesiviruses with features distinct from both VESV and FCV in phylogeny, serology and cell culture specificities. CaCV is a probable species in the Vesivirus genus, as stated by ICTV. It is still unclassified to date and the evidence presented herein should facilitate the classification and acceptance of CaCV as a species of vesivirus.
Many viruses found in human and other animal species can also infect dogs asymptomatically or cause respiratory, digestive, neurologic and genital diseases with mild to severe symptoms. In response to the use of dogs in military services and laboratory studies, etiological studies of canine diseases were conducted in 1963–1978 at the Walter Reed Army Institute of Research (WRAIR) [3, 4]. In addition to several known canine viral pathogens [5, 6], four unidentified viruses were recovered in Walter Reed Canine Cells (WRCC) producing similar cytopathic effects (CPE). The isolates were not recognized by available human and dog reference virus antisera. Studies of their physicochemical properties and electron microscope observations identified the isolates as likely caliciviruses. Our recent whole genome sequencing of these canine isolates clearly identified them as vesiviruses and elucidated their genetic relationships to the other members of the Caliciviridae family. We herein report the viral isolation and characterization results, which were made in 1963–1978 canine diseases etiological study but were not published, and additional genomics analysis supporting the serological diversity of CaCV strongly suggesting that these isolates and similar CaCV are a unique species within Vesivirus genus [7–9].
Senecavirus A (SVA), a member of the family Picornaviridae, genus Senecavirus, is a recently identified single-stranded RNA virus closely related to members of the Cardiovirus genus [1, 2]. SVA was originally identified as a cell culture contaminant and was not associated with disease until 2007 when it was first observed in pigs with Idiopathic Vesicular Disease (IVD) [2, 3]. Vesicular disease is sporadically observed in swine, is not debilitating, but is significant due to its resemblance to foreign animal diseases, such as foot-and-mouth disease (FMD), whose presence would be economically devastating to the United States [3, 4]. IVD disrupts swine production until foreign animal diseases can be ruled out. Identification and characterization of SVA as a cause of IVD will help to quickly rule out infection by foreign animal vesicular disease pathogens.
IVD in association with SVA has been observed recently in Canada, the United States, and Brazil, in the absence of other vesicular foreign animal diseases [3, 5, 6]. A quick test to diagnose SVA infection is necessary to help rule out infection by foreign animal diseases without prolonged disruption of animal movement. As of now, SVA infection is diagnosed by RT-PCR, a serum neutralizing assay, indirect fluorescent antibody test (IFA), or competitive enzyme-linked immunosorbent assay (cELISA) [6–9]. RT-PCR is a rapid method to determine if animals are acutely infected with virus or if vesicles contain virus, but a negative result cannot be used to rule out previous herd exposure since clinical signs of infection are usually resolved within 1–2 weeks [6, 10]. Presence of antibodies to SVA may indicate previous infection and possible presence of the virus in a herd. Although serum neutralization and IFA test for the presence of serum antibodies, ELISA is more rapid and convenient. A rapid, specific and sensitive assay for the detection of SVA-specific antibodies is needed. A cELISA for the detection of SVA antibodies is available, but requires an antibody competition between well characterized monoclonal antibodies and serum antibodies for binding to inactivated viral antigen. An indirect ELISA only requires a purified antigen and so is not susceptible to mutations that change reactivity of the monoclonal antibody-binding epitope. An SVA VP1 ELISA has recently been used to examine antibody presence in sows and piglets naturally infected with SVA, however a comprehensive validation of this assay was not shown. Although numerous ELISA kits used for the detection of viral antibodies are commercially available, an indirect ELISA kit is not yet commercially available for the detection of anti-SVA antibodies in pigs.
An optimized, well characterized, quick and inexpensive indirect ELISA for the detection of SVA antibodies as well as a thorough examination of antibodies and their levels over a time course following infection is needed. The aim of this study was to develop and characterize an indirect ELISA assay to identify serum antibodies to SVA as well as examine the kinetics of the presence and levels of SVA antibodies over a time course following infection. The SVA VP2 ELISA developed in this study can now be used to help differentially diagnose IVD due to SVA, helping to quickly rule out the presence of an economically devastating foreign animal disease.
Viral infections are of major concern within the pig production industry, in that they cause not only severe disease but also subclinical infections that can have severe economic consequences. It is becoming evident that a number of factors, including multiple microorganisms, often act synergistically to create a certain clinical picture. This is particularly evident in, for example, complex respiratory and enteric diseases. Known major respiratory viruses include porcine reproductive and respiratory syndrome virus (PRRSV), swine Influenza A virus (swine AIV), and pseudorabies virus (PRV); porcine respiratory coronavirus (PRCV) and porcine circovirus type 2 (PCV2) are also believed to be involved. It is highly plausible that other viruses could be of importance as well, but they have been overlooked. Some reasons for this could be that diagnostic labs are not searching actively for them, because they are not believed to be involved in respiratory diseases, or because they have not even been discovered yet. Additionally, it is known that the combination of viruses and/or bacteria, as well as different management and environmental factors, are of importance. A viral infection that under certain circumstances does not cause any apparent problems for the infected host can, under other circumstances, have severe consequences. Considering this, it is important not only to identify individual viruses in a host but also to investigate the entire viral community.
High-throughput sequencing (HTS) combined with metagenomic approaches has been shown to be a powerful tool for elucidating the aetiology behind diseases, as well as identifying novel viral species from humans, animals, and plants. For pigs, many novel viruses have been identified, including porcine circovirus type 3 (PCV3), porcine bocavirus (PBoV), and atypical porcine pestivirus (APPV). The role that many of these viruses potentially play in disease development is yet to be determined. Apart from detecting previously unrecognised viruses, HTS and metagenomics is a valuable tool for investigating the viral community.
In this study, viral metagenomics was used to characterise the viral community of individual pigs coming from a conventional herd with respiratory lesions at slaughter with the aim of identifying possible agents connected with this disease complex. To understand complex diseases and the role that different viruses may or may not play, we need to identify which viruses are circulating in the porcine population regardless of health status. Thereby, we could possibly identify viruses that make up a basal virome at different ages, in different herds, in connection to different health statuses, etc. This knowledge could be used to understand the effects of different viral-coinfections. In pursuit of this goal, we also investigated the virome of specific pathogen-free (SPF) pigs.
Since the coincidental isolation of Seneca Valley virus (SVV), recently termed Senecavirus A (SV-A), as a cell culture media contaminant in 2002, a number of serologically similar viruses were identified and grouped to the classification of Senecavirus. The primary sequence analysis of the conserved polypeptide regions (P1, 2C, 3C and 3D) of the first isolate (SVV-001) showed that the virus is most closely related to cardioviruses in the family of Picornaviridae. The single-stranded RNA genome of SV-A displays the secondary structural features of an internal ribosome entry site (IRES) that resembles the IRES element of classical swine fever virus (CSFV) of the family Flaviviridae, giving rise to the possibility that genetic exchange may have occurred between members of Picornaviridae and Flaviviridae during persistent co-infection in pigs. Importantly, SV-A is a natural oncolytic agent, with the ability to selectively replicate in; and kill human tumor cells of neuroendocrine origin, thus, the virus is being advanced as a tool for potential therapeutic intervention of cancer.
Swine are considered to be the natural hosts of SV-A and all known SV-A sequenced isolates have been obtained from pigs. Previously, by regression analysis of partial genome sequences, it was suggested that different isolates of SV-A had a common ancestor and were assumed to have been introduced into the US pig populations (http://www.europic.org.uk/Europic2006/posters/Knowles.svv.01.pdf). Virus isolated in cell culture from tissue specimens of a diseased pig presenting vesicular lesions on the snout and feet in 2005, was identified by the National Veterinary Services Laboratories’ (NVSL) Foreign Animal Disease Diagnostic Laboratory (FADDL) as SV-A using a broad pan-viral microarray (unpublished data). More recently, this vesicular disease syndrome, with as yet unidentified etiology, has been termed swine idiopathic vesicular disease (SIVD) [5, 6]. Despite the isolation of SV-A in cell culture, FADDL has been unsuccessful at reproducing clinical signs by experimental inoculation of pigs with live virus. Negative observations were also made by other laboratories who conducted animal inoculations with multiple SV-A isolates. Singh et al (2012) proposed SV-A as the causative agent of SIVD from a detailed clinical, diagnostic and histopathological study on a Chester White boar suffering from anorexia, lethargy, lameness and vesicular lesions. However, association of SV-A with SIVD, or as the sole causative agent, is speculative at this time since the virus has also been isolated from pigs lacking clinical disease. SIVD has been reported in pigs in the continents of North America and Australia [6, 9–11]. Although SIVD itself does not pose an economic concern, veterinary diagnosis from clinical signs is complicated since similar vesicular lesions can be formed due to common viral infections such as parvovirus, enterovirus, toxins in food supply, or burns [12–16]. Additionally, SIVD clinically resembles high consequence transboundary animal diseases (TADs) such as foot and mouth disease (FMD), swine vesicular disease (SVD), vesicular stomatitis (VS), and vesicular exanthema of swine (VES). A few laboratory methods have been developed for detection of SV-A including a virus serum antibody neutralizing test and a competitive enzyme linked immunosorbant assay (cELISA), which are not widely available [7, 17]. The principal aim of this study was to develop a specific real-time RT-PCR (RT-qPCR) assay for fast, sensitive, and quantitative detection of SV-A RNA in vesicular diagnostic tissues.
AIV, avian influenza virus; HA, hemagglutinin; IAV, influenza A virus; NA, neuraminidase; NCR, non-coding regions; vRNA, viral RNA; vRNP, viral ribonucleoprotein; VSV, vesicular stomatitis virus.
Swine influenza is an infectious disease caused by RNA viruses which are highly contagious. Multiple strains of this virus are common throughout pig populations worldwide and cause significant economic losses in industry. At least three SwIV subtypes H1N1, H1N2 and H3N2 are currently circulating in the swine population despite regular vaccinations, and exchange of influenza viruses between human and swine is common and not a one-way street,. The traditional vaccine is less effective because it cannot possibly include all the strains actively infecting people in the world. Therefore, therapeutic alternatives for preventing infections and maintaining the health of livestock are highly warranted.
Probiotics are defined as live microorganisms which when administered in adequate amounts confer a health benefit on the “host” (FAO/WHO, 2001). Although targeting the intestinal tract probiotics can also affect mucosal defence in general, including immune responses in the respiratory tissues,. Probiotic bacteria, as a part of gut microflora, are reported to promote the host defense and to modulate the immune system. Probiotics have been recently shown to mediate antiviral effects against certain viruses in vitro and in vivo
,,, and the effect of various strains of probiotics on the course of virus infections in pigs is being studied intensively. However, while some descriptive information on the effect of probiotics on model viruses such as vesicular stomatitis virus (VSV), transmissible gastroenteritis virus (TGEV) and rotaviruses,,, are available, no such data are yet available for swine influenza viruses which are most important in view of their exquisite zoonotic capacity. It is commonly believed that the mammalian influenza viruses are restricted to the respiratory tissue and thus may hardly be affected by probiotics acting in the intestine. However, a recent report on the pathogenesis of seasonal influenza virus H1N1 in ferrets shows that this virus is also present in the intestine. Furthermore it is world acknowledged that avian influenza viruses frequently infect in the intestine of the avian host. Therefore it appears justified to include influenza viruses in studies on the probiotic inhibition of virus multiplication both in vitro and in vivo.
Enterococcus faecium NCIMB 10415 is authorized in the EU for safe use as a probiotic feed additive and therefore represents a suitable probiotic to study its possible anti-viral properties. We had previously carried out experiments with this probiotic in the context of bacterial infection which showed that E. faecium modulates intestinal immunity in piglets,. In the present study we explored if E. faecium affects the replication of swine influenza virus H1N1 and H3N2 in a macrophage (3D4/21) and epithelial cell line (MDBK).
The evolution of emerging diseases is associated with factors embedded in the concept “host-agent-environment triangle” (1). To infect the host and cause disease, the pathogen needs to evade host defenses, which may occur through single point mutations, genome rearrangements, recombination and/or translocation (2). Genetic uniformity generated through genetic selection of the host (3) and the fact that demographic changes, intensification of farming, and international commerce have occurred markedly over the last decades, must be also considered as essential factors for the development of emerging diseases (4–6).
As well as in humans, emerging diseases drastically affect animal populations, especially food-producing animals. Livestock production in large communities (i.e., pig farms or poultry flocks) represents an excellent environment to facilitate the transmission and maintenance of huge viral populations, contributing to the pathogen evolution (through mutation, recombination and reassortment, followed by natural selection) (7–9). The intensification of livestock during the last four decades has probably been one of the main factors that contributed to the emergence of new pathogens and/or pathogen variants, leading to changes in the epidemiology and presentation of diseases (10).
The number of viral infectious diseases in swine has significantly increased in the last 30 years. Several important worldwide distributed viruses have been reported in this period, including Porcine reproductive and respiratory syndrome virus (PRRSV, family Arteriviridae), Porcine circovirus 2 (PCV-2, family Circoviridae) and Porcine epidemic diarrhea virus (PEDV, family Coronaviridae). In addition to those worldwide widespread viruses, an important number of novel swine pathogens causing different types of diseases has been described (11, 12). Although their economic impact might be variable, they are considered significant infection agents and their monitoring is nowadays performed in some parts of the world. Among others, relevant examples are Porcine deltacoronavirus (associated with diarrhea) (12), Senecavirus A (causing a vesicular disease and increased pre-weaning mortality) (11), Porcine sapelovirus (found in cases of polioencephalomyelitis) (13), Porcine orthoreovirus (assumed to cause diarrhea) (14), Atypical porcine pestivirus (cause of congenital tremors type II) (15) and HKU2-related coronavirus of bat origin (associated with a fatal swine acute diarrhea syndrome) (16).
Besides overt emerging diseases of swine, many other novel infectious agents have been detected in both healthy and diseased animals, and their importance is under discussion. This group of agents is mainly represented by Torque teno sus viruses, Porcine bocavirus, Porcine torovirus and Porcine kobuvirus, which are thought to cause subclinical infections with no defined impact on production (13, 17, 18). An exception may be represented by Hepatitis E virus (HEV); although it seems fairly innocuous for pigs, it is considered an important zoonotic agent (19, 20). Recently, a novel member of the Circoviridae family named Porcine circovirus 3 (PCV- 3), with unknown effects on pigs, has been discovered (21, 22).
Porcine circovirus 3 (PCV-3) was first described in 2015 in North Carolina (USA) in a farm that experienced increased mortality and a decrease in the conception rate (21). Sows presented clinical signs compatible with porcine dermatitis and nephropathy syndrome (PDNS) and reproductive failure. In order to identify the etiological pathogen, aborted fetuses and organs from the affected sows were collected for further analyses. Whilst histological results were consistent with PCV-2-systemic disease, both immunohistochemistry (IHC) and quantitative PCR (qPCR) methods to detect PCV-2 yielded negative results. Samples were also negative for PRRSV and Influenza A virus. Homogenized tissues from sows with PDNS-like lesions and three fetuses were tested through metagenomic analysis, revealing the presence of an uncharacterized virus (21). Further analyses using rolling circle amplification (RCA) followed by Sanger sequencing showed a circular genome of 2,000 nucleotides. Palinski et al. (21) also performed a brief retrospective study through qPCR on serum samples from animals clinically affected by PDNS-like lesions (but negative for PCV-2 by IHC) and pigs with porcine respiratory diseases. Results revealed PCV-3 qPCR positivity in 93.75 and 12.5% of the analyzed samples, respectively (21).
Interestingly, almost concomitantly, another research group from the USA reported a clinical picture pathologically characterized by multi-systemic and cardiac inflammation of unknown etiology in three pigs of different ages ranging between 3 and 9 week-old (22). Several tissues from these animals were tested by next-generation sequencing (NGS) methods and PCV-3 genome was found. Beyond NGS, in situ hybridization was performed in one out of these three pigs, confirming PCV-3 mRNA in the myocardium (cytoplasm of myocardiocytes and inflammatory cells mainly, although to a very low frequency).
Based on these two initial works, the name PCV-3 was proposed as the third species of circoviruses affecting pigs, since pairwise analysis demonstrated significant divergence with the existing PCVs. The novel sequences showed < 70% of identity in the predicted whole genome and capsid protein amino acid (aa) sequence compared to the other members of the Circovirus genus (22). Taking into account the economic importance and the well-known effects of PCV-2 on the swine industry, a new member of the same family like PCV-3 should not be neglected. Studies on epidemiology, pathogenesis, immunity and diagnosis are guaranteed in the next few years, but the scientific community is still in its very beginning on the knowledge of this new infectious agent. Therefore, the objective of the present review is to update the current knowledge and forecast future trends on PCV-3.
Swine Transmissible Gastroenteritis Coronavirus (TGEV), as a member of the coronaviridae, is a kind of single-stranded RNA virus, which produces villous atrophy and enteritis, leading to the serious financial loss to the whole pig industry. The traditional detection methods, including virus isolation, virus immunodiagnostic assays and PCR tests have the shortcomings, such as precise instruments requirement, elaborate result analysis demand, high cost, long detection time and so forth, which prevent these methods from being widely used[1-4]. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method, which amplifies DNA/RNA with high specificity, sensitivity and rapidity under isothermal condition. It has already found wide application in RNA virus detection, such as Foot-and-mouth Disease Virus, Swine Vesicular Disease Virus, Taura Syndrome Virus, Severe Acute Respiratory Syndrome Coronavirus and H5N1 Avian Influenza Virus[9,10]. In this study, LAMP method was applied in developing qualitative and quantitative detection system of TGEV, while its specificity and sensitivity were assessed.
Seneca Valley virus (SVV) is a single-stranded, positive-sense RNA virus belonging to the species Senecavirus A in the genus Senecavirus in the family Picornaviridae [1, 2]. Although the species name Senecavirus A has been used in some publications as the virus name with an acronym of SVA, in fact the virus name is Seneca Valley virus. The SVV genome (approximately 7.3 kb) contains a single open reading frame (ORF) flanked by a long 5′ untranslated region (UTR; 668 nucleotides) and a short 3′ UTR (68 nucleotides) followed by a poly(A) tail. The polyprotein translated from the single ORF is predicted to be post-translationally processed into four structural proteins (VP4, VP2, VP3, and VP1) and seven nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C, and 3D).
SVV was initially incidentally identified as a contaminant in PER.C6 cell cultures in 2002. From 1988 to 2001, a number of virus isolates were sporadically recovered from pigs in various U.S. states but with no detailed description of the clinical symptoms. Sequence analysis of these retrospective virus isolates suggested that these viruses were the same as SVV. Thereafter, SVV was sporadically identified in pigs with swine idiopathic vesicular disease in Canada in 2007 and in the U.S. in 2010, but not much attention was drawn to this virus. At the end of 2014 and the beginning of 2015, multiple outbreaks of vesicular disease in weaned and adult pigs as well as increasing mortality rates of neonatal piglets (1–4 days of age) were reported in Brazil [6–8]. SVV was consistently detected from the pigs with vesicular lesions while other vesicular viral pathogens were not detected. Starting from July 2015, SVV was consistently detected from increasing swine idiopathic vesicular disease cases observed in exhibition, commercial finisher, and breeding swine herds in the U.S.. Foreign animal disease investigations indicated that other vesicular viral pathogens, such as foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), vesicular stomatitis virus (VSV), and vesicular exanthema of swine virus (VESV), were negative in these cases. Subsequently, SVV detection was reported by other laboratories in the U.S. [11–16], China [17–21], Canada, Thailand, and Colombia. Vesicular lesions were induced in pigs following experimental inoculation with the contemporary U.S. isolates of SVV [25, 26], confirming that SVV is a vesicular viral pathogen. In one experimental infection study, a historical SVV isolate (SVV-001) did not cause overt clinical diseases in the inoculated pigs but it established infection in pigs and induced an immune response. Since the vesicular lesions caused by SVV infection are clinically indistinguishable from those caused by other vesicular disease viruses (e.g., FMDV, SVDV, VSV, and VESV), differential diagnosis is mandatory. RT-PCR is a sensitive and fast method commonly used to differentiate vesicular viral pathogens.
A number of SVV specific gel-based (conventional) RT-PCR, nested RT-PCR, real-time RT-PCR (rRT-PCR), reverse transcription droplet digital PCR, and loop-mediated isothermal amplification assays have been described in the literatures although not all of them have been fully validated [6, 28–35]. Compared to the conventional RT-PCR assays, rRT-PCR is generally more sensitive and suitable for high throughput testing with shorter turnaround time. It is noteworthy that conduction of rRT-PCR assays requires trained technicians and expensive instruments; rRT-PCR assays are mainly performed in the laboratory rather than for on-site applications.
In recent years, a fluorescent hydrolysis probe-based insulated isothermal PCR (iiPCR) technology has been described. The iiPCR and RT-iiPCR can be used for the detection of DNA and RNA molecules. The principle of the iiPCR is to amplify the DNA/RNA by cycling the reaction components through different temperature gradients (denaturation, annealing, and extension) in a capillary tube on a simple Nucleic Acid Analyzer [36, 37]. The iiPCR technology and a commercially available field-deployable device (POCKIT™ combo system), which includes a taco™ mini Automatic Nucleic Acid Extraction System and a POCKIT™ Nucleic Acid Analyzer (GeneReach USA, Lexington, MA, USA), allow automatic detection and interpretation of PCR results within 1–1.5 h. It has been shown that iiPCR or RT-iiPCR assays have excellent sensitivity and specificity for the detection of various targets, including swine pathogens, such as classical swine fever virus (CSFV), FMDV, porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and porcine reproductive and respiratory syndrome virus (PRRSV) [38–41], and various pathogens in shrimp, dogs, cats, poultry, ruminants, and horses [42–52].
In the present study, a SVV rRT-PCR targeting the conserved 5′ UTR and a SVV RT-iiPCR targeting the conserved 3D gene region were developed and validated for the detection of SVV RNA.
Foot-and-mouth disease (FMD) is a highly contagious vesicular disease of cloven-hoofed animals posing a great threat to the world economy. The disease is caused by FMD virus (FMDV), a highly divergent small RNA virus of the genus Aphthovirus under the family Picornaviridae. There are 7 serotypes of the virus namely O, A, C, Asia-1, SAT-1, SAT-2 and SAT-3. Infection with one serotype does not produce immunity to other serotypes. Among domesticated animals, cattle, buffalo, swine, sheep and goat are susceptible to the disease. African wild buffaloes maintain SAT serotypes of the virus in oropharyngeal region and act as carriers to cloven-hoofed wildlife. The disease is transmitted via contaminated air and fomites or direct contact with infected animal. After infection, the virus replicates rapidly, and viremia occurs within a day. The virus transmission occurs at 0.3-0.7 day before the appearance of clinical signs. Generally, FMD is characterized by formation of vesicles on the feet, buccal mucosa and mammary glands, and drooling of saliva in the form of string. After recovery, the affected animals retain the virus in their oro-pharynx and may act as a carrier for the disease transmission to the susceptible animals.
The FMDV is 25 nm in diameter and consists of a single-stranded positive-sense RNA genome surrounded by four structural proteins to form an icosahedral capsid. FMD viral genome consists of a large single open reading frame (ORF) flanked by highly structured 5’ and 3’untranslated regions (UTR). The 5’ UTR is divided into five elements, S-fragment, poly C tract, pseudo-knots, cis-acting replicative element (cre), and internal ribosome entry site (IRES). The IRES serves for internal initiation of protein synthesis in cap-independent manner. The 3’ UTR contains a short stretch of RNA which folds into a specific stem-loop structure of about 90 nucleotides, followed by a poly(A) tract of variable length. Both the UTRs play roles in virus translation and RNA replication. The ORF is translated to single polyprotein, which is subsequently processed by virus-encoded proteases (e.g. 3Cpro) to produce four structural and eight non-structural proteins. These proteins self-assemble to form icosahedral, virus-like particles (VLPs), which contain 60 copies of each structural protein (VP1, VP2, VP3, and VP4) encapsidating the single stranded RNA genome.
The disease is endemic and prevalent in many countries in Africa, the Middle East, Asia and South America, and occurs in the form of outbreaks. In India, the disease is caused by the serotypes O, A, and Asia-1 among which the serotype O is the most prevalent (93.3%). Currently, the disease is controlled by vaccination in developing countries while developed countries follow stamping out policy. Inactivated whole virus vaccine has been widely used to control and prevent the disease. However, the preparation of the inactivated virus vaccine requires costly biocontainment facilities, large quantities of live virus and cold chain maintenance, and is associated with risk of escape of the live virus from the vaccine manufacturing unit to the environment and from vaccine due to incomplete activation, and is also not suitable for differentiating infected from vaccinated animals (DIVA) strategy.
Various approaches have been tried to develop alternatives to the inactivated whole virus vaccine. VLPs based vaccine has received a lot of attention and considered one of the most appealing approach for contemporary vaccine design due to their immunogenic properties and high safety profile for vaccine delivery platforms. VLPs have been generated by use of various expression systems like vaccinia virus, adenovirus, Escherichia coli and baculovirus. VLPs of the FMDV have been produced in vitro by co-expression of P1-2A and 3Cpro. These are structurally similar to whole virus particles but noninfectious and safe, induce efficient humoral and cellular immune response and also suitable for DIVA strategy.
Recombinant adenoviruses have become vectors of choice for target gene delivery, expression of foreign antigens, and have been used in gene therapy, vaccination and cancer therapy. The adenoviruses are considered as powerful vectors because of their ability to recombine in culture, high production titers, relatively high capacity for transgene insertion, efficient transduction of both quiescent and actively dividing cells, and for inducing humoral and cellular immune responses. FMD molecular vaccine based on replication deficient human adenovirus serotype 5 (hAd5) carrying FMD capsid genes was developed and licensed for use as emergency response tool during any FMD outbreak in the USA. The hAd5 carrying FMDV capsid protein antigen (P1-2A) along with 3Cpro have been tested in mice, guinea pigs, swine and cattle to protect them from homologous challenge virus. The hAd5 containing full length 2B has been reported to induce a rapid and increased FMDV-specific humoral and cellular immune responses as compared to the original vector.
Hence, considering the above facts, this study was carried out to construct and characterize hAd5 expressing capsid proteins (P1-2A) along with full-length 2B, 3B and 3Cpro (wild-type: 3Cwt and/mutant type: 3Cm) of the FMDV O//IND/R2/75.
Vaccine development against infectious diseases has classically been based on live attenuated or inactivated infectious agents. Recently, the approach of vaccination with recombinantly expressed antigens and immunogens from viral and non-viral delivery systems has been introduced to the repertoire. In this context, immunization with surface proteins and antigens has elicited strong humoral and cellular immune responses and vaccinated animals showed protection against challenges with lethal doses of infectious agents or tumor cells.
The types of non-viral vectors applied include liposomes, immunostimulatory complexes (ISCOMs) composed of adjuvant Quil A and peptides, and multiple antigen peptides (MAPs) also known as dendrimers. A number of viral vectors based on adenoviruses, alphaviruses, avipoxiviruses, enteroviruses, flaviviruses, measles viruses (MV), rhabdoviruses, and vaccinia viruses have been engineered for vaccine development. In this context, self-replicating RNA virus vectors have proven highly efficient for immunization studies in various animal models. Among RNA viruses, rabies virus (RABV) and vesicular stomatitis virus (VSV) belonging to the rhabdovirus family carry a single-stranded RNA (ssRNA) genome of a negative polarity. Likewise, MV possess a negative-sense ssRNA genome. In contrast, flaviviruses and alphaviruses are of positive polarity. West Nile virus and Kunjin virus are the most common flaviviruses applied for immunization studies. Similarly, expression vectors have been engineered for alphaviruses such as Semliki Forest virus (SFV), Sindbis virus (SIN) and Venezuelan equine encephalitis virus (VEE).
In this review, various self-replicating RNA virus vectors are described and their applications as recombinant virus particles, RNA replicons and layered DNA plasmids are compared. Moreover, examples are given of utilization of self-replicating RNA virus systems for immunization studies in various animal models to elicit humoral and cellular immune responses and to generate neutralizing antibodies, as well as protection against challenges with pathogens and tumor cells. Finally, a summary of clinical trials already conducted or in progress that apply self-replicating RNA viruses is presented. However, due to the large number of publications available, it is only possible to present key findings and examples of vaccine development for self-replicating viral vectors.
Multisystemic inflammation and myocarditis were initially linked with the presence of PCV-3 (22). One single study described PCV-3 in weaned pigs that suffered from gastro-intestinal disorders (diarrhea), showing higher prevalence in pigs with clinical signs (17.14%, 6 out of 35) compared to those with non-diarrhea signs (2.86%; 1 out of 35) (87). In another report, animals with congenital tremors were analyzed and PCV-3 was the only pathogen found in the brain, with high amount of viral DNA (101).
Comparison of negative samples and positive samples from the infection time course showed that ELISA and IFA were correlated (n = 231, p < 0.0001) (Fig. 5). The IFA has a specificity of 100% and a sensitivity of 90.3%. Agreement on negative samples was 89% and agreement from 4 to 60 days after appearance of clinical signs varied from 73 to 100%. However, on the day that clinical signs were first observed (Day 0), test agreement was 40%, which was due to samples testing ELISA positive, but IFA negative or suspect (Fig. 5). In cases of disagreement, ELISA was usually positive while IFA was negative or suspect. Three percent of total samples were ELISA negative, but IFA positive (days 11, 18, and 60).