Aleutian Disease Virus Infection Research Articles

Beyond Rabies: Are Free-Ranging Skunks (Mephitis mephitis) in British Columbia Reservoirs of Emerging Infection?

Wild animal reservoirs are an important source of emerging and zoonotic infection. Skunks (Mephitis mephitis) are a reservoir of skunk strain rabies virus in Canada, with the exception of some areas including the province of British Columbia (BC). Beyond rabies, the reservoir status of skunks for emerging and zoonotic pathogens in BC is unknown. From March 2011 to February 2015, 50 free-ranging skunks were necropsied and tested for 4 pathogens: influenza A, Aleutian disease virus (ADV), Leptospira spp. and Salmonella spp. Two skunks (4%) with respiratory disease caused by influenza A (H1N1) pdm09 were detected during the human flu season suggesting that skunks may represent a target population for reverse zoonosis of this strain of influenza A virus. High prevalence of ADV infection was detected (43/50, 86%). Two of the infected skunks exhibited Aleutian disease (AD) suggesting that skunks act as both a reservoir and a target population for the virus. Most studies of ADV have focused on the potential for infection of free-ranging species living near mink farms. Our study suggests that urban skunks may be a primary host for the virus independent of domestic mink. Whether skunks act as a reservoir of ADV infection for other peridomestic species will depend on host specificity of the viral strains. Leptospira interrogans was detected in 18% (9/49) of the skunks. Identification of the serovar(s) detected is needed to determine any public health risk of leptospirosis following exposure to infected skunks. Salmonella spp. was isolated from three of 43 skunks (7%), specifically S. Typhimurium, S. Muenchen and S. Enteritidis. These serotypes cause disease in humans, but the low prevalence of infection suggests there is a low risk for zoonotic transmission.

Detection of Aleutian disease virus by loop-mediated isothermal amplification.

In this study, a loop-mediated isothermal amplification (LAMP) assay was developed and optimized for the detection of Aleutian disease virus (ADV) in minks. The amplification could be completed within 45min under isothermal condition by employing a set of six ADV genome-specific primers. The amplification results could be visualized directly with the naked eye by using fluorescent dye. Comparative experiments showed that the LAMP assay is superior to conventional polymerase chain reaction for the detection of both experimental and field samples. Results of current study indicated that the LAMP assay is a rapid and reliable technique for routine diagnosis of ADV infection in minks.

Infection with Aleutian disease virus-like virus in a captive striped skunk

A 5-month-old captive female striped skunk (Mephitis mephitis) was evaluated because of lethargy, signs of depression, azotemia, and erythema of the skin around the eyes. Antemortem diagnostic tests revealed renal disease but failed to identify an etiologic agent. A diagnosis of severe nonsuppurative interstitial nephritis was made on the basis of results of histologic examination of renal biopsy specimens. The skunk was administered isotonic fluids SC daily and later every other day because of the handling-related stress. Because of the skunk's deteriorating condition, it was euthanized after 24 days of supportive care. Aleutian disease was diagnosed on the basis of positive results of a PCR assay that targeted the DNA from Aleutian disease virus (ADV); positive results for ADV were also obtained by use of plasma counterimmunoelectrophoresis and an ELISA. Genetic sequencing of the 365-base pair PCR product revealed 90% sequence identity with mink ADV. In the skunk of this report, infection with a skunk-specific parvovirus resulted in clinical signs and pathologic changes similar to those associated with ADV infection in mink. For skunks with signs of renal failure, differential diagnoses should include parvovirus infection. In confirmed cases of infection with this ADV-like virus, appropriate quarantine and biosecurity measures should be in place to prevent spread to other susceptible animals within a zoological collection.

DNA vaccination with the Aleutian mink disease virus NS1 gene confers partial protection against disease

Aleutian disease virus (ADV) causes severe losses in mink. This happens in nature as well as in farms. In spite of several attempts to provide an efficient protective protein based vaccine, experiments have failed so far. Only partial protection has been obtained. The aim of this work was to construct and test a protective DNA vaccine based on the gene encoding for the ADV non-structural protein 1 (NS1) and to test this construct as a potential vaccine candidate against ADV infection or disease. First, the vaccine construct was tested by in vitro transfection studies. NS1 protein expression was found by immunofluorescent studies and the expected size of translated protein confirmed by Western blot. Then, 18 female mink were divided into three groups: a control group, a DNA vaccinated group, and a group which received DNA vaccine plus a boost with recombinant NS1 protein in the last immunization. After virus challenge, the two DNA vaccinated groups induced higher antibody levels in the first 23 weeks of the 32 week observation period. One month after virus challenge, the most interesting finding was, that the “DNA + protein” group exhibited a significantly higher percentage of CD8 + cells, when compared to the levels in the two other groups. This, we believe, indicate a memory CTL response created by the vaccination. Most CD8 + cells were found to contain interferon gamma as measured by FACS intracellular staining. Severity of Aleutian disease was judged by quantification of plasma gammaglobulin levels and mink death statistics. The findings let us to conclude, that the two DNA vaccinated groups of mink did show milder disease characteristics, but that the vaccine effect also in this trial could only be characterized as partial.

Mink infected with aleutian disease virus have an elevated level of CD8-positive T-lymphocytes

Lymphocytes, monocytes, granulocytes, B-lymphocytes and CD8-positive T-lymphocytes of non-infected mink and mink infected with Aleutian disease virus (ADV) were measured by flow cytometry. The gammaglobulin levels of the sera were also measured. Besides development of hypergammaglobulinaemia in the infected mink, the most pronounced finding was that the number of CD8-positive lymphocytes doubled on average during development of Aleutian disease, while the number of B-lymphocytes did not change dramatically. The enhanced CD8 frequency was still apparent 6 months after initial ADV infection of the mink. The present experiments contribute to a better understanding of the immune deficiency stage seen in mink infected with ADV.

Passive transfer of antiviral antibodies restricts replication of Aleutian mink disease parvovirus in vivo

When mink kits were infected neonatally with a highly virulent strain of Aleutian disease virus (ADV), 100% of both Aleutian and non-Aleutian genotype mink died of interstitial pneumonia characterized by permissive ADV infection of alveolar type II cells. Treatment of infected kits with either mink anti-ADV gamma globulin or mouse monoclonal antibodies against ADV structural proteins reduced mortality by 50 to 75% and drastically reduced the severity of clinical signs. Interestingly, mink kits that survived the acute pulmonary disease all developed the chronic form of immune complex-mediated Aleutian disease. Thus, the antibodies directed against ADV structural proteins were capable of modulating the in vivo pathogenicity from an acute fulminant disease to a chronic immune complex-mediated disorder. The mechanism of this modulation was examined by strand-specific in situ hybridization. We found that the number of ADV-infected type II cells was the same in both untreated and antibody-treated kits. However, in the treated kits, viral replication and transcription were restricted at the cellular level. These data suggested that antibodies prevented acute viral pneumonia by restricting the intracellular level of viral replication and that the relevant antigenic determinants were contained within the viral structural proteins. The restricted levels of viral replication and transcription seen in antibody-treated mink kits resembled the levels observed in infected adult mink and suggested a role of antiviral antibodies in development of persistent infection and chronic immune complex disease.

Immunoglobulin classes of Aleutian disease virus antibody.

Aleutian disease virus (ADV) persistently infects mink and causes marked hypergammaglobulinemia. Immunoglobulin class-specific antisera were used to define the total immunoglobulin of each class by radial immunodiffusion and the immunoglobulin class of ADV-specific antibody by immunofluorescence in experimentally and naturally infected mink. Electrophoretic gamma globulin closely reflects the immunoglobulin G (IgG) level in mink, and the majority of the increased immunoglobulin and ADV antibody in infected mink is IgG. IgM becomes elevated within 6 days after infection, reaches peak levels by 15 to 18 days, and returns to normal by 60 days after infection. The first ADV antibody demonstrable is IgM, and most mink have virus-specific IgM antibody for at least 85 days postinfection. Serum IgA levels in normal mink are not normally distributed, and ADV infection causes a marked elevation of IgA. Low levels of ADV-specific IgA antibody can be shown throughout the course of infection. Failure of large amounts of virus-specific IgG antibody to inhibit the reaction of virus-specific IgM and IgA antibodies suggests that the various classes of antibodies are directed against spatially different antigenic determinants. The IgM and IgA were shown not to be rheumatoid factors.

Characteristics of inapparent Aleutian disease virus infection in mink

Inapparent of nonprogressive Aleutian disease virus (ADV) infection is a subclinical but persistent virus infection of mink. Mink with the inapparent type of ADV infection when subjected to stress did not develop the progessive form of the disease. However, when challenged with a large dose of the virus, these mink did develop progressive Aleutian disease indicating that they were not highly resistant to the virus. Sera of mink with either the progressive of the inapparent type of ADV infection did not neutralise the virus. The anti-ADV antibody activity in mink with inapparent type of ADV infection was in the IgG fraction of the serum the same as in mink with progressive Aleutian disease. These data indicate that the resistance of the mink with inapparent infection as compared to mink with progressive Aleutian disease was not due to a difference in the class of immunoglobulin response to the virus. However, mink with progressive Aleutian disease showed a greatly increased immunoglobulin response.