75 Flaviviridae
The family name of the Flaviviridae (Latin flavus, yellow) is derived from yellow fever, a disease of humans caused by a flavivirus, with jaundice as a major clinical feature. Members of the family are 40 to 60 nm in diameter with icosahedral capsids and tightly adherent envelopes which contain either two or three virus-encoded proteins, depending on the genus. The genome is composed of positive-sense single-stranded RNA. Replication of virus occurs in the cytoplasm with maturation in cytoplasmic vesicles and release by exocytosis. The mature virions, which are generally labile, are sensitive to heat, detergents and organic solvents. The family comprises four genera. Two genera, Flavivirus and Pestivirus, contain viruses of veterinary importance. The genus Flavivirus contains more than 50 members assigned to several serologically defined groups. Most members of the genus are arboviruses, which require either mosquitoes or ticks as vectors. The genus Pestivirus contains viruses of veterinary importance, namely bovine viral diarrhoea virus 1 and 2, border disease virus and classical swine fever virus. The sole member of the Hepacivirus genus, hepatitis C virus, causes hepatitis in humans. The genus Pegivirus contains viruses isolated from primates (pegivirus A, which is also known as hepatitis G virus) and from fruit bats (pegivirus B).
Clinical infections
In the genera Flavivirus and Pestivirus there are several viruses of particular veterinary importance (Table 75.1). Four members of the genus Flavivirus, louping ill virus, Japanese encephalitis virus, Israel turkey meningoencephalitis virus and Wesselsbron virus, cause disease in domestic animals. In addition, infection with West Nile virus, an important human pathogen, causes fatal disease in birds and horses. Other members of the genus which are important human pathogens include yellow fever virus, dengue virus, Japanese encephalitis virus, tick-borne encephalitis virus and St Louis encephalitis virus.
Table 75.1 Viruses of veterinary importance in the genera Flavivirus and Pestivirus.
| Genus | Virus | Hosts | Comments |
| Flavivirus | Louping ill virus | Sheep, cattle, horses, red grouse, humans | Present in defined regions of Europe. Transmitted by the tick Ixodes ricinus; produces encephalitis in sheep and other species |
| Japanese encephalitis virus | Waterfowl, pigs, horses, humans | Widely distributed in Asia. Transmitted by mosquitoes. Waterfowl are reservoir hosts. Infection in pigs results in abortion and neonatal mortality | |
| Wesselsbron virus | Sheep | Occurs in parts of sub-Saharan Africa. Transmitted by mosquitoes. Produces generalized infection, hepatitis and abortion | |
| Israel turkey meningo- encephalomyelitis virus | Turkeys | Reported in Israel and South Africa. Transmitted by mosquitoes. Progressive paresis and paralysis | |
| West Nile virus | Birds, humans, horses | Birds are the natural hosts. Transmitted by mosquitoes. Serious nervous disease reported sporadically in humans and horses. Has become widespread in North America since first appearance in 1999 | |
| Pestivirus | Bovine viral diarrhoea virus 1 and 2 | Cattle (sheep, pigs) | Occur worldwide. Cause inapparent infection, bovine viral diarrhoea and mucosal disease. Congenital infection may result in abortion, congenital defects and persistent infection due to immunotolerance |
| Border disease virus | Sheep | Occurs worldwide. Infection of pregnant ewes may result in abortion, congenital abnormalities or persistent infection | |
| Classical swine fever (hog cholera) virus | Pigs | Highly contagious, economically important disease with high mortality. Generalized infection with nervous signs and abortion; congenital tremors in piglets |
The four recognized members of the Pestivirus genus which infect domestic species are closely related antigenically. Infections with these pestiviruses may be inapparent, acute or persistent and are economically important worldwide. An additional four viruses have been proposed but are not yet officially recognized, namely atypical pestivirus (isolates from cattle, sheep and pigs, also referred to as HoBi-like virus), Bungowannah virus, pronghorn antelope pestivirus and giraffe pestivirus.
Bovine viral diarrhoea and mucosal disease
Infection with bovine viral diarrhoea virus (BVDV) is common in cattle populations throughout the world. The virus can cause both acute disease, bovine viral diarrhoea (BVD), and a protracted form of illness, mucosal disease, arising from persistent infection. Using cell culture, cytopathic and non-cytopathic biotypes are recognized. The biotype most often isolated from cattle populations is non-cytopathic. Cytopathic isolates can arise from non-cytopathic BVDV as a result of recombination events, including incorporation of host RNA and duplication of viral RNA sequences in the NS2-3 gene resulting in cleavage of NS2-3 and increased production of NS3. Two genotypes, now considered separate species, BVDV 1 (classical BVDV isolates) and BVDV 2 (atypical BVDV isolates), are recognized on the basis of differences in the 5′-untranslated region of the viral genome. Both genotypes contain cytopathic and non-cytopathic isolates and produce similar clinical syndromes in cattle. However, only type 2 isolates have been associated with thrombocytopenia and a haemorrhagic syndrome, first described in North America.
Persistently infected (PI) animals, which shed virus in secretions and excretions, are particularly important sources of infection. Persistent infection develops when infection of the foetus with a non-cytopathic strain occurs before day 120 of gestation. About 1% of animals in an infected population are persistently infected and viraemic. The presence of cattle with persistent infection in a herd results in constant exposure of the other cattle to virus, producing a high level of herd immunity. The outcome of transplacental spread depends on the age of the foetus at the time of infection. During the first 30 days of gestation, infection may result in embryonic death with return of the dam to oestrus. The effects of foetal infection between 30 and 90 days of gestation include abortion, mummification and congenital abnormalities of the CNS, often cerebellar hypoplasia. Foetuses which become infected after day 120 of gestation can mount an active immune response and are usually normal at birth. If virus invades the foetus before development of immune competence, immunotolerance to the agent results, with persistent infection for the lifetime of the animal. The virus involved in this persistent infection is non-cytopathic. Later, usually between six months and two years of age, a cytopathic biotype emerges as a consequence of mutation of the non-cytopathic virus or of recombination with nucleic acid of the host cell or other non-cytopathic biotypes. Cytopathic isolates have a particular tropism for gut-associated lymphoid tissues. These molecular changes in BVDV may lead to the development of mucosal disease in some animals.
Most BVDV infections are subclinical. Outbreaks of BVD are usually associated with high morbidity and low mortality. When present, clinical signs include inappetence, depression, fever and diarrhoea. Although a significant proportion of PI animals are clinically normal, some are born undersized and exhibit growth retardation and decreased viability. Mucosal disease is usually sporadic in occurrence. Clinical signs include depression, fever, profuse watery diarrhoea, nasal discharge, salivation and lameness. Ulcerative lesions are present in the mouth and interdigital clefts. The case fatality rate is 100%.
A tentative diagnosis may be possible on the basis of clinical signs and pathological findings. Laboratory confirmation requires demonstration of antibody, viral antigen or viral RNA. Seroconversion and the presence of viraemic animals are necessary for confirmation of established infection in a herd. Most losses arising from BVDV infections in herds result from the effects of prenatal infections and mucosal disease. Control strategies are directed at preventing infections which can lead to the birth of PI animals. Killed, attenuated live and temperature-sensitive mutant virus vaccines have been developed. The elimination of BVDV from a herd requires the identification and removal of PI animals. Testing to detect viral antigen or viral RNA in blood or ear notch samples is used to identify PI animals. In national eradication programmes, herds containing PI animals can be detected by testing bulk milk or pooled blood samples for antibodies to BVDV.
Border disease
This congenital disease of lambs, also known as hairy shaker disease, occurs worldwide. Border disease, which was first reported from the Welsh–English border, is caused by infection of the foetus with a pestivirus which is non-cytopathic. Pestivirus isolates from sheep can infect other domestic ruminants and pigs. Moreover, pestivirus isolates from a number of domestic species can infect pregnant sheep causing border disease in their offspring.
Persistently infected animals shed virus continuously in excretions and secretions. These animals tend to have a low survival rate under field conditions, although some may survive for several years without developing clinical signs. Persistently infected ewes may give birth to persistently infected lambs. Acute infections in susceptible sheep are transient and result in immunity to challenge with homologous strains of border disease virus (BDV).
Virus is probably acquired by the oronasal route. In susceptible pregnant ewes, infection results in placentitis and invasion of the foetus. The immune response of the ewe does not protect the developing foetus. The age of the foetus at the time of infection ultimately determines the outcome. The foetus develops immune competence between 60 and 80 days of gestation. Foetal death may follow infection prior to the development of immune competence, the outcome being resorption, abortion or mummification. Foetuses which survive infection become immunotolerant and remain persistently infected. These animals may be clinically normal at birth or may display tremors and hairy birthcoat, consequences of viral interference with organogenesis. Congenital defects in affected lambs include skeletal growth retardation, hypomyelinogenesis and enlarged primary hair follicles with reduced numbers of secondary follicles. Infection after day 80 of gestation induces an immune response with elimination of the virus and the birth of a healthy lamb. Foetal infection during mid-gestation when the immune system is developing may result in lesions in the CNS including cerebral cavitation and cerebellar dysplasia.
In flocks infected with BDV, there may be an increase in the number of abortions and weak neonatal lambs. Characteristic signs of infection in newborn lambs include altered body conformation, changes in fleece quality and tremors. Affected lambs are often small and their survival rate is poor. In well-nursed lambs, the neurological signs may gradually abate and such animals may eventually become clinically normal.
The characteristic clinical signs are diagnostic. Dysmyelination may be demonstrable histologically in the CNS. Immunocytochemical staining can be used to demonstrate virus in brain tissue. Serological testing, employing methods such as serum neutralization and ELISA, can be used to determine the extent of infection in a flock.
Control should be based on identification and removal of persistently infected animals and precautions to avoid introduction of infected animals into a flock. Where such a policy is not feasible, breeding stock should be deliberately mixed with persistently infected animals at least two months before mating.
Classical swine fever (hog cholera)
This highly contagious, potentially fatal OIE-listed disease of pigs, although still present in many countries, has been eradicated from North America, Australia and most European countries. Infected animals occur in the wild boar populations in Europe, which act as reservoirs of infection. Direct contact between infected and susceptible animals is the main means of transmission. In endemic areas, the disease is spread principally by movement of infected pigs. Shedding of virus may begin before clinical signs become evident. Virulent virus is shed in all excretions and secretions. Virus strains of moderate virulence may result in chronic infection with continuous or intermittent shedding by infected pigs. In addition, congenital infections with strains of low virulence may result in the birth of persistently-infected piglets. Spread between premises can occur indirectly, particularly in regions with a high density of pig farms. The virus, which is relatively fragile and does not persist in the environment, is not spread over long distances by air movement. However, it can be transmitted mechanically by personnel, vehicles and biting arthropods. Despite its lability, classical swine fever virus (CSFV) can survive for long periods in protein-rich biological materials such as meat or body fluids, particularly if chilled or frozen. Although legislation is in place in most European countries prohibiting the feeding of uncooked swill, outbreaks of classical swine fever can still be traced to waste food fed to pigs.
Pigs are usually infected by the oronasal route. The tonsil is the primary site of viral multiplication. Virus spreads to regional lymph nodes and viraemia develops after further viral multiplication. Virus, which has an affinity for vascular endothelium and reticuloendothelial cells, can be isolated from all major organs and tissues. In acute swine fever, vascular damage in conjunction with severe thrombocytopenia results in widespread petechial haemorrhages. A non-suppurative encephalitis with prominent perivascular cuffing is present in most CSFV-infected pigs. Virus strains of reduced virulence can cause a mild form of the disease. In pregnant sows, infection may result in stillbirths, weak newborn piglets with congenital tremors and, occasionally, clinically normal piglets.
Following an incubation period of up to 10 days, affected animals develop high fever and become inappetent and depressed. Sick pigs are inclined to huddle together. Vomiting and constipation are followed by diarrhoea. Some animals may die soon after developing convulsions. A swaying gait usually precedes posterior paresis. Most cases of acute classical swine fever succumb within 20 days after infection. Signs of disease are milder in infections caused by strains of low virulence.
Although clinical signs and history may provide evidence for a tentative diagnosis, laboratory confirmation is essential, particularly with infections caused by strains of reduced virulence. In acute disease, haemorrhages are present in many internal organs and on serosal surfaces. Petechiae are often present on kidney surfaces and in lymph nodes. Other gross pathological features of diagnostic significance are splenic infarction and ‘button’ ulcers in the mucosa of the terminal ileum near the ileocaecal valve. Rapid confirmation is possible using direct immunofluorescence on frozen sections of tonsillar tissue, kidney, spleen, distal ileum and lymph nodes. Antigen-capture ELISAs are available commercially and suitable for detection of viral antigen in blood or organ suspensions. The RT-PCR assays for detection of CSFV RNA are sensitive and rapid, replacing most other methods used for virus detection. Serological testing is useful on farms infected with strains of low virulence or for serological surveys.
The disease is notifiable in many countries which have adopted slaughter policies and banned vaccination. Pigs and pig products should not be imported from countries where infection with CSFV is present. Swill must be boiled before being fed to pigs. In countries where the disease is endemic or during the early stages of an eradication programme, vaccination may be used. Live vaccines attenuated either by serial passage in rabbits or in tissue culture are currently used. These vaccines are safe and effective. The use of recombinant E2 glycoprotein marker vaccine in conjunction with a specific ELISA capable of detecting antibodies to the other main envelope glycoprotein, Erns, offers a means of distinguishing vaccinated from naturally infected pigs.
Louping ill
The name ‘louping ill’ derives from the Scottish vernacular for ‘leaping’ or ‘bounding’, an allusion to the abnormal gait of some affected animals. Although primarily a disease of sheep, louping ill can occur in other animals and also in humans. The disease, which is largely confined to Britain and Ireland, has also been described in Norway, Spain, Bulgaria and Turkey. Louping ill virus is transmitted by the tick Ixodes ricinus and the seasonal incidence and regional distribution of the disease reflect periods of tick activity in suitable habitats such as upland grazing. The host range of I. ricinus is wide and infection with louping ill virus can occur in many vertebrate species including sheep, cattle, horses, deer, red grouse and humans. On farms where infection is endemic, losses occur mainly in sheep under two years of age. Following infection, most sheep acquire life-long immunity. Young lambs are protected by colostral antibody.
Viral replication occurs initially in lymph nodes draining sites of inoculation. Viraemia follows, with dissemination to other lymphatic organs and to the brain and spinal cord. The speed and onset of the immune response are important in preventing spread of virus and limiting the degree of damage in the central nervous system. Immunosuppression caused by infection with Anaplasma phagocytophilum, the agent of tickborne fever, is considered to be responsible for increased mortality in sheep with louping ill.
A history of neurological signs or unexplained deaths in sheep in endemic areas during periods of tick activity may be indicative of louping ill. Laboratory confirmation is usually required. A non-suppurative encephalomyelitis is usually detectable histologically. Detection of louping ill virus using an RT-PCR protocol has been described. Antibody to the virus can be detected using complement fixation and gel diffusion tests. Inactivated vaccines are protective. Purchased sheep or animals within the flock intended for breeding should be vaccinated at six to 12 months of age. Colostral immunity usually protects lambs for the first six months of life. Measures aimed at clearing grazing land of tick habitats and dipping of sheep decrease the risk of infection with louping ill virus.
West Nile virus
West Nile virus (WNV), a member of the Japanese encephalitis virus serocomplex, is a mosquito-borne flavivirus which infects many species of animals including horses, birds and humans. From its initial detection in New York in 1999, the virus has spread throughout North America and to Central and South America. Mosquito vectors include Culex species and also Aedes and Anopheles species. The virus is transmitted in enzootic cycles involving mosquitoes and birds. Incidental infections occur in humans and domestic animals. Transmission occurs through the bite of insect vectors and horizontal transmission is not described in domestic animals. Migrating birds may carry the virus to new geographical regions. Although some birds may remain asymptomatic, many species including crows, ravens, jays and geese develop high levels of viraemia with high mortality. A proportion of infected horses and humans develop clinical signs. Neurological signs in horses include leg weakness and flaccid paralysis. Virus can be recovered from a wide range of tissues in avian species but brain and spinal cord are the preferred specimens for laboratory diagnosis of disease in horses. Virus isolation can be carried out in cell culture. Infection can be confirmed by detection of viral antigen by immunoassay and immunohistochemistry or by detection of viral RNA by RT-PCR. Suitable serological tests include ELISA and plaque reduction neutralization test. Control is based on vaccination and commercially available vaccines for horses currently include an inactivated whole virus vaccine and a recombinant canarypox-vectored vaccine.