In 1946 Olafson and associates described gastroenteritis with severe diarrhea in dairy herds in New York state. Some affected animals developed ulcers in the nasal and oral mucosa. Abortions were also seen. Although morbidity rates seemed high, mortality rates were low. Bacteria were not found in blood or tissues that produced the same clinical symptoms in healthy cattle. Through transmission and immunity studies, the ailment was differentiated from rinderpest, which has similar symptoms. The new disease was termed bovine viral diarrhea.

Seven years later mucosal disease (MD) was first described by Ramsey and Chivers in beef and dairy cattle of varying ages in Iowa and neighboring states. Similar to Olafson, they saw symptoms of ulcerative mucosal lesions and diarrhea. Mortality rates were high. It was thought to be a separate disease entity from viral diarrhea. Again, no bacterial agent was found to be associated with the disease. Cross-immunity studies in cattle and cell culture methodologies eventually led researchers to realize that the same virus caused bovine viral diarrhea and MD, and that MD is a possible sequela to BVD infection.

In the 1960s and 1970s it was learned that bovine viral diarrhea virus (BVDV) is closely related to the hog cholera virus and the ovine border disease virus. All three are in the viral family known as Flaviviridae and belong to the genus Pestivirus.

Today BVDV infections are seen in all ages of cattle throughout the world and has significant economic impact due to productive and reproductive losses. In the last two decades, recent advances in molecular genetic research has led to an increased understanding of the wide range of clinical diseases associated with BVDV. Learning how to control the spread of the virus is a challenge that still faces researchers.

PATHOGENESIS OF BVDV INFECTIONS
Pathogenesis of acute infections with BVDV
Acute infection of immunocompetent cattle with BVDV can result in a wide range of clinical syndromes. Although not entirely clear, the outcome of an acute infection is probably related to several factors including strain of virus, age of host, immune and physiologic status of the host, and the presence of other pathologic agents.
The majority of acute BVDV infections are caused by noncytopathic viruses. Cattle acutely or persistently infected with BVDV are the primary source of virus. Infected animals shed virus in nasal and oral secretions, feces and urine. The primary virus entrance route is probably oral nasally. Other less important routes of entry may include infected semen, biting insects, and contaminated instruments. Following entry and contact with the mucosal lining of the mouth or nose, initial replication occurs in epithelial cells with a predilection for the palatine tonsils. From here, the virus is able to spread systemically through the blood stream. Spread can occur through both free virus in the serum and virus infected leucocytes, particularly lymphocytes and monocytes. Isolation of virus from serum or leucocytes is generally possible between 3 and 10 days post infection. During systemic spread, the virus is able to gain entry to most tissues with a preference for lymphoid tissues. However, the tissues infected may vary between different virus strains.
Following acute infection, it is generally accepted that most clinical outcomes are mild. Mild fevers, diarrhea, and leukopenia have most commonly been described following acute infection. However, some viral strains have been associated with much more severe disease including fatal hemorrhagic diarrhea and fatal thrombocytopenia. The pathogenesis of these more severe clinical outcomes are still unclear. Secondary infection by other pathogens may occur because of various degrees of immune system dysfunction created by acute BVDV infections. The major role that BVDV plays in shipping fever is thought to be that of local immunosuppression in the lungs, allowing for secondary bacterial infections to occur.
Finally, acute infections with BVDV are important in pregnant cattle because of the ability of the virus to cross the placenta and cause intrauterine infections of the fetus. Fetal infections can result in early embryonic death, abortion, congenital defects, or the birth of calves persistently infected with BVDV.

Pathogenesis of intrauterine infections
It is well documented that BVDV can cross the placenta and infect fetuses of all ages. The outcome of these infections are largely dependent on the stage of gestation when infection occurs.
Embryos appear to be resistant to infection until they hatch from the zona pellucida. This occurs around day 10 of gestation. Susceptibility becomes greatest following implantation which occurs at day 20 of gestation. During this early phase of pregnancy, infection with BVDV often result in early embryonic death and resorption of the fetus.
Fetal infection during the first trimester of gestation can result in abortion or the development of a mummified fetus. If a fetus survives this early infection, they invariably become persistently infected with BVDV. Persistent infections develop when BVDV is circulating around 100 days of gestation. At this stage of gestation, immunocompetence is being established in the fetus. With immunocompetence comes recognition of self antigen, a process necessary to prevent autoimmunity. If BVDV virus is present during this time, it too is recognized as self and allowed to survive and replicate in the fetus and the post natal animal. In most cases, persistently infected animals do not perform well, often dieing in utero or soon after birth. However those that do survive remain infected for life.
Fetal BVDV infection during the late first, second and early third trimester of gestation may also result in the formation of several different types of congenital anomalies. The stage of fetal development determines the type of defect that occurs. The most common defect is cerebellar hypoplasia. Other defects that have been described include cataracts, retinal degeneration and hypoplasia, optic neuritis, skeletal malformations, hypotrichosis, and general growth retardation. The mechanisms of these defects are not known. Possible explanations include direct cell damage by replicating virus or indirect cell damage by response of the developing immune system to the virus.
As the immune system develops during the second trimester of gestation, the fetus is able to mount an immune response to a BVDV infection. However virus may still damage the fetus resulting in congenital defects and/or abortion. Infection late in pregnancy usually results in the birth of clinically normal calves. Calves infected after the development of the immune system are born with precolostral antibodies to BVDV.

Pathogenesis of mucosal disease
Mucosal disease is the classic disease syndrome that people often associate with BVDV. Until recently, the pathogenesis of mucosal disease was unknown. However, with advances in molecular biology techniques, the mystery of mucosal disease is being solved.
Mucosal disease in nature is probably a rare event because of the special circumstances that are required for it to occur. The first factor that must be present for mucosal disease to occur is that an animal must be persistently infected with BVDV. This persistent infection occurs when a fetus becomes infected in utero between 1-4 months of gestation with noncytopathic BVDV. Persistent infections with cytopathic BVDV have never been documented. At this stage of gestation, immunocompetence is being established in the fetus. With immunocompetence comes recognition of self antigens. If BVDV virus is present during this time, it too is recognized as self and allowed to survive and replicate in the fetus and the postnatal animal. In most cases, persistently infected animals do not perform well, often dieing in utero or soon after birth. However those that do survive remain infected for life. Virus is allowed to replicate unchallenged and is continuously shed into the environment. The host mounts no immune response against the original virus. However, if the host becomes infected with another BVD virus which is antigenically different from the persistent virus, an immune response can occur to the second virus.
The second factor needed for mucosal disease to occur is the superinfection of the persistently infected animal with an antigenically similar cytopathic BVDV. This can occur in several ways. The most common occurrences is for a mutation to occur at specific sites in the noncytopathic virus genome. This mutation may occur in several different ways including insertion of RNA into or deletion of RNA from the noncytopathic BVDV genome. The end result of the mutation is that a new protein, termed p80, is produced during translation of the cytopathic BVDV RNA genome. This protein is present in noncytopathic BVDV as part of a larger protein termed p125. The role that p80 plays in causing cytopathology is not understood. It should be noted that the mutation does not change the antigenic makeup of the virus, therefor the cytopathic virus is not recognized by the host's immune system and is allowed to replicate without challenge. Other sources of cytopathic viruses would include modified live vaccines or experimental challenge. Antigenic homology between the cytopathic and noncytopathic virus must be maintained for mucosal disease to occur.
Although the mechanisms of cellular damage are unclear, the replicating cytopathic BVDV results in rapid depletion of the gut-associated lymphoid tissue (peyers patches) with subsequent necrosis of the gastrointestinal mucosa. Severe diarrhea ensues which eventually leads to the animal's death.
Recently, a form of mucosal disease called chronic mucosal disease has been described. The same factors are needed for this syndrome to evolve. A cow persistently infected with noncytopathic BVDV is superinfected with cytopathic BVDV. However, instead of being antigenically identical to the original virus, slight differences exist in the antigenic makeup of the new cytopathic virus. This allows the persistently infected cow to mount an immune response against the cytopathic virus. However, the immune response is often incomplete and pathology slowly evolves leading to the eventual death of the animal.

Type I and Type II BVDV
The genetic and antigenic diversity between different strains of BVDV is quite varied. However, it has become clear that two major groups of virus exist which differ significantly with respect to both their genetic and antigenic makeup. These two groups are referred to type I BVDV and type II BVDV. Although differences exist at the molecular level, the major aspects of the pathogenesis of disease caused by type I and type II BVDV appear to be the same. The major difference is that acute infections with type II BVDV have been associated with more severe clinical syndromes. The reasons for this increase in severity are unknown.
Molecular Biology
Bovine viral diarrhea virus is a member of the genus Pestivirus within the flaviviridae family of virus. Hog cholera virus and border disease virus in sheep are also members of the Pestivirus genus. Representatives of other genera in the flaviviridae family include hepatitis C virus, yellow fever virus and dengue virus.
The genome of BVDV consists of a single strand of positive sense RNA which is about 12,300 nucleotides long. A single open reading frame exists which is approximately 3900 codons long. The open reading frame is flanked by 5' and 3' untranslated regions which are important for the initiation of translation and RNA stability. The open reading frame is translated into a single polyprotein which is then processed by both viral and cellular enzymes into mature viral proteins.
The BVDV genome encodes for both structural and nonstructural proteins. The structural proteins include the capsid protein C (p14) and three glycoproteins Erns, E1, and E2 (gp48, gp25, and gp53). [Note: protein names given are proposed new nomenclature; names in parenthesis are original nomenclature.] The capsid protein functions to package the genomic RNA and to provide structure for the formation of the virion envelope. The three glycoproteins are associated with the lipid envelope. The E2 protein contains the major recognition sites for the production of neutralizing antibodies against BVDV by infected or vaccinated cattle. The genetic sequence which codes for part of the E2 protein is extremely variable between strains of BVDV which probably reflects selective pressure against these neutralizing sites by the immune system.
Six nonstructural proteins are encoded by the noncytopathic BVDV genome. Npro (p20) is the first protein produced from the open reading frame. It has papain-like protease activities. The next nonstructural protein produced is NS23 (p125). This protein has several unique characteristics which suggest its involvement in multiple functions. These characteristics include a very hydrophobic domain, a zinc-finger, a protease, and a helicase. Cytopathic BVDV strains have changes within the coding region for p125 which result in the production of the protein NS3 (p80). This protein is unique to the cytopathic BVDV biotype. The NS3 protein of cytopathic BVDV contains the protease and helicase activity of the NS23 protein. Other nonstructural proteins include NS4A (p10), NS4B (p32), and NS5A (p58). Knowledge of these proteins are limited. However they appear to be important in viral replication. The final protein produced is NS5B (p75) which is thought to be the RNA-dependent-RNA polymerase needed to replicate the viral genome.
Replication of BVDV begins with receptor mediated endocytosis into a cell. The E2 glycoprotein appears to mediate this step. Once in the cell, the viral RNA is released into the cytosol. RNA translation then begins with the 5' untranslated region serving as an internal ribosomal entry site. Viral proteins can be detected as early as three hours after cell infection.
Following gene translation, the large polyprotein product is processed by both cellular and viral enzymes into mature proteins. Once the RNA-dependent RNA polymerase is produced, new genomic RNA is produced to be packaged into virus packages. Viral packaging occurs in either the Golgi apparatus or endoplasmic reticulum where they acquire their lipid envelope through budding into the vesicle lumen. Mature virus packages are then released from the cell exocytosis. New virus can be released as early as 10 hours post cell infection.


PREVENTION AND CONTROL OF BVDV
Due to the complex pathogenesis and insidious nature of BVDV infections in the cattle population, the laboratory diagnosis is an essential component of developing measures for the control and prevention of BVDV infections. The positive isolation of BVDV from animal tissue submissions or from aborted fetuses, should provide a strong indication for further epidemiological investigations within the identified infected herds. In these situations, the ultimate goal should be the establishment of sound control and prevention methods by prevention of any potential exposure to BVDV (ie. removal of persistently infected animals and preventing the introduction of infected animals) and by protecting animals by using effective vaccination programs.

Laboratory Diagnosis

• Acute Infections

The diagnosis of acute infections must be done during the window of opportunity for virus isolation, as early as 3 days postinfection to 8 or 10 days postinfection. Some animals may be virus isolation positive for only 2 to 3 days during the course of BVDV infection. A whole blood sample is the best sample to collect and submit for BVDV isolation to identify animals acutely infected. A viremia would be detectable in serum by virus isolation during a more narrow window of opportunity than from whole blood. In addition, swabs from mucosal or nasal surfaces can be collected and submitted for virus isolation. Since nucleic acid methods are not influenced by the development of neutralizing antibodies, these methods may remain positive for longer periods during the convalescent stage when neutralizing antibody would interfere with virus isolation. In acute outbreaks in dairy herds, the collection of samples from other herdmates in addition to the severely affected animals may aid in the ability to make positive diagnoses. Whole blood and clotted blood samples should be collected from any animals with mild diarrhea, slight increase in temperature, decrease in feed consumption, or decrease in milk production. Virus isolation should be done on whole blood samples and the serum stored for submission with a paired sample 30 days later for serology. To properly asses serology, paired acute and convalescent samples collected 30 days apart are required to identify four fold increases in serum antibody titers following convalescence. Specimens collected at postmortem examinations from lymphoid organs as mentioned previously should be submitted for virus isolation from any animals that die or fetuses from any abortions that may occur. Many BVDV associated abortions are virus isolation negative. Some aborted fetuses may already have produced antibodies, the presence of which will confirm intrauterine infection. Maternal serology is only seldom helpful because seroconversion has often taken place before the abortion. If the dam on the other hand is antibody negative BVDV can be ruled out as the cause of abortion. Most BVDV associated congenital defects occurring following infection after the onset of immunological competence and therefore calves with these defects have BVDV antibody. The diagnosis of BVDV induced congenital defects in calves should included both virus isolation and serology to detect BVDV-specific antibody prior to uptake of colostrum.

• Persistent infections

The identification of persistently infected animals is most routinely done by virus isolation. The level of viremia in persistently infected animals is generally quite high (106 CCID50/ml of serum) but may vary from 102 to 107 CCID50/ml. In addition, the level of viremia may decline in individual animals over time. In most cases, for routine identification of persistently infected animals, serum is adequate for virus isolation. In young calves, maternal antibody will decrease the level of free virus in serum and virus isolation may be falsely negative. Due to colostral antibody, in young calves less than 3 months of age the best sample remains to be whole blood in which the mononuclear cells are separated for virus isolation. It has been suggested that serology can be used to identify seronegative animals as candidates for testing by virus isolation to identify persistent infections. Although persistently infected animals are immunotolerant, they may develop neutralizing antibody titers to BVDV. Therefore, serology should not be used as a screening method to identify animals to test for virus isolation. Since the number of persistently infected animals in any one herd will generally be low, any potentially infected animals should not be excluded from being tested. Persistent infections should only be determined by identification of BVDV by virus isolation in sequential samples collected 30 days apart. By testing the animal 30 days apart it is possible at the same time to test for a four-fold increase in antibody titer should the first virus isolation have been due to acute infection.

HERD SCREENING

• Indications

If a laboratory diagnosis of BVDV infection has been made from any submitted clinical samples, then there is an indication and an obligation for further investigation to be conducted at the herd level. Currently, herd screening involves individual animal testing by virus isolation which requires a certain amount of time and expense. Therefore, herd screening should not be done unless a commitment is made to establish and continue long term plans for the control and prevention of BVDV. If possible, due to the time and expense of herd screening, a positive laboratory diagnosis should be sought to indicate the infection status of a herd prior to herd screening.
The infection status of a herd may be established by additional methods such as testing bulk milk for anti-BVDV antibody or the testing of the bulk milk sample for BVDV by RT-PCR amplification. These types of assays are best utilized as an initial screening test to identify herds in which further diagnostic testing should be done to identify the source of virus spread (ie. persistently-infected animals). In some situations it is difficult to convince herd owners that the expense of herd testing is warranted. Therefore, positive identification of BVDV by preliminary PCR screening from a bulk milk sample would give good justification for further herd testing by virus isolation. Although the PCR bulk milk test could not be used to rule out BVDV infection, a positive result would provide useful information. Positive PCR results would be weighted more heavily than negative results. Therefore, the use of this assay may be most beneficial as a method of focusing on or identifying BVDV positive herds for development of control strategies and not as a definitive test to ensure that a herd is negative for BVDV. Other screening methods such as serology on groups of nonvaccinated calves at 6 months of age also are useful as discussed previously.

• Methods

The most common approach to herd screening would be to obtain serum samples from all the animals in the herd over 3 months of age. In addition, whole blood samples should be collected from calves less than 3 months of age. Basically, all animals in the herd should be tested; therefore pregnant animal must be considered as 2 animals. Virus isolation using a microtiter immunoperoxidase detection methods is the most common method used for testing such large numbers of samples. Using this method, results from 2 serial passages are generally available within 5 to 9 days. In addition, any calves born for the next 9 months must be tested to ensure that no additional persistently infected animals are born that were in utero at the time of testing. Gestating animals may be convalescent from an acute infection and virus isolation negative while a persistently infected fetus remains in utero. Due to the interference of maternal antibody, it is recommended that calves be retested at 3 to 4 months of age, prior to vaccination, to ensure that no persistently infected animals would be advanced and placed in the replacement heifer breeding groups. During the 9 to 12 month period of testing, segregation of age groups and prevention of exposure of young replacements by preventing direct contact and controlling traffic of personnel and equipment with any breeding females is essential to prevent the cycle of fetal exposure and infection.

• Interpretation

In some cases following herd testing, no persistently infected animals are identified. In these herds the persistently infected animals may have been culled or have died previously. Another possible explanation is that the herd infection with the particular strain of BVDV may have likely resulted in abortions than the production of persistently infected calves. In any case, it is essential to establish that no persistently infected animals are present in the herd and that the cycle of maternal-fetal transmission is broken. In these herds, it is important that calf testing be continued and that the importance of management decisions are stressed which reflect sound control and prevention measures to prevent exposure to BVDV. If persistently infected animals are identified these animals should be culled and removed from the herd. The potential exposure of pregnant animals to any of the identified persistently infected animals should be noted for future screening of new born animals.

• Vaccination

Due to the high prevelance of BVDV in the cattle population it is manditory that BVDV vaccination be done due decrease potential losses due to BVDV infection. The use of killed or modified-live vaccines can provide protection by decreasing the consequences of acute infections. However, it is questionable whether killed or modified-live vaccines provide complete fetal protection from the development of in utero fetal infections.

 

Source: vetmed