Temporal replication of the Pullman strain of Aleutian disease virus in royal pastel mink.

Information was sought on the temporal replication of Aleutian disease virus in 27 royal pastel mink. Groups of three were examined 8 to 126 days after they were inoculated subcutaneously with 10(3) 50% lethal doses of the Pullman strain. Much individual variation was noted in the onset of infection, occurrence of viremia, and extent of virus replication in the tissues. Thus, virus was detected in lymph nodes regional to the site of inoculation in only some mink during the first 14 days after inoculation. During this period, virus was often present as well in the mesenteric lymph node and spleen. First detected on day 10, viremia was present in all mink examined on day 28 but occurred irregularly thereafter, even when virus was widespread in the tissues. Except in five mink succumbing to the disease, the tissue distribution of virus after day 28 tended to be more limited, and the titers were generally lower than they had been earlier. Even though present in the lymph nodes and spleen, virus was often absent from the kidney, liver, and intestine after day 28. Specific antibody was detected on day 28 and was present in all mink thereafter, ostensibly without any adverse effect on virus replication. In most mink, the infection was considered subclinical, for it was usually not accompanied by a rise in serum gamma globulin or by morphologic evidence of the disease. The virologic findings in this study have a bearing on the relationship of subclinical infections to both horizontal and vertical transmission of the virus.     

Demonstration of Aleutian disease virus-specific lymphocyte response in mink with progressive Aleutian disease: comparison of sapphire and pastel mink infected with different virus strains

Lymphocyte blastogenesis was used to study the antiviral lymphocyte response of sapphire (Aleutian) and pastel (nonAleutian) mink inoculated with Pullman or Utah 1 Aleutian disease virus (ADV). Both mink genotypes developed a virus-specific response when inoculated with Utah 1 ADV. In contrast, after inoculation of Pullman ADV, sapphire mink had a positive virus-specific response, whereas pastel mink did not. Response occurred late after infection (8 wk) and correlated with the development of progressive Aleutian disease (AD). The response to keyhole limpet hemocyanin (KLH) and concanavalin A (Con A) was also determined. Most mink of either genotype, inoculated with either virus strain, maintained an anti-KLH response during disease. Most mink also responded to Con A, although some exhibited suppressed Con A response late in the disease course. These results indicated that mink develop an anti-ADV lymphocyte response during progressive AD and are not immunosuppressed with regard to other antigens or mitogens.     

Interferon response in normal and Aleutian disease virus-infected mink.

Studies were done to determine whether differences in interferon production are responsible for the resistance of pastel mink to Aleutian disease. The abilities of normal pastel and sapphire mink to produce interferon when inoculated with either Newcastle disease virus or a synthetic polyribonucleotide, poly (I):poly (C), were identical, even to the production of a novel, acid-labile interferon. The resistance of pastel mink to Aleutian disease did not correlate with interferon production, because neither sapphire nor pastel mink produced detectable amounts of interferon when infected with either the Pullman strain of Aleutian disease virus (ADV) or the highly virulent Utah I strain. Sapphire mink infected with the Pullman strain responded normally to poly (I):poly (C) early in the course of the disease, but interferon production was impaired late, when the mink were hypergammaglobulinemic and had renal, vascular, and hepatic lesions. These data suggest that ADV Pullman neither stimulates nor interferes with interferon production in infected mink and may represent a mechanism whereby ADV can more readily establish infection.     

Temporal distribution of transmissible mink encephalopathy virus in mink inoculated subcutaneously.

Information was sought on the temporal distribution of transmissible mink encephalopathy virus in royal pastel mink inoculated subcutaneously with 10(3.0) 50% intracerebral lethal doses of the Idaho strain. As determined by intracerebral assay in mink, extremely little replication of the virus occurred during the preclinical stage of infection. It seemed largely limited to lymph nodes draining the site of inoculation. Virus first appeared in the central nervous system (CNS) at 20 weeks, when all mink were still clinically normal. Early spongiform degeneration, limited to the posterior sigmoid gyrus of the frontal cortex, was first found at 28 weeks, or a few weeks before onset of clinical disease in most of the mink. Once virus reached the CNS, where greater concentrations occurred than elsewhere, it appeared in many extraneural sites (spleen, liver, kidney, intestine, mesenteric lymph node, and submandibular salivary gland). These seemingly anomalous findings, especially the limited extraneural replication of virus as a prelude to infection of the CNS, suggest that mink are not natural hosts of the virus. The results of this study support the generally held view that transmissible mink encephalopathy arises from chance or inadvertent infection of ranch mink with an exogenous virus, most likely feed-borne wild scrapie virus.     

Characterization of chimeric full-length molecular clones of Aleutian mink disease parvovirus (ADV): identification of a determinant governing replication of ADV in cell culture.

The ADV-G strain of Aleutian mink disease parvovirus (ADV) is nonpathogenic for mink but replicates permissively in cell culture, whereas the ADV-Utah 1 strain is highly pathogenic for mink but replicates poorly in cell culture. In order to relate these phenotypic differences to primary genomic features, we constructed a series of chimeric plasmids between a full-length replication-competent molecular clone of ADV-G and subgenomic clones of ADV-Utah 1 representing map units (MU) 15 to 88. After transfection of the plasmids into cell culture and serial passage of cell lysates, we determined that substitution of several segments of the ADV-Utah 1 genome (MU 15 to 54 and 65 to 73) within an infectious ADV-G plasmid did not impair the ability of these constructs to yield infectious virus in vitro. Like ADV-G, the viruses derived from these replication-competent clones caused neither detectable viremia 10 days after inoculation nor any evidence of Aleutian disease in adult mink. On the other hand, other chimeric plasmids were incapable of yielding infectious virus and were therefore replication defective in vitro. The MU 54 to 65 EcoRI-EcoRV fragment of ADV-Utah 1 was the minimal segment capable of rendering ADV-G replication defective. Substitution of the ADV-G EcoRI-EcoRV fragment into a replication-defective clone restored replication competence, indicating that this 0.53-kb portion of the genome, wholly located within shared coding sequences for the capsid proteins VP1 and VP2, contained a determinant that governs replication in cell culture. When cultures of cells were studied 5 days after transfection with replication-defective clones, rescue of dimeric replicative form DNA and single-stranded progeny DNA could not be demonstrated. This defect could not be complemented by cotransfection with a replication-competent construction.     

Replication of Aleutian mink disease parvovirus in vivo is influenced by residues in the VP2 protein.

Aleutian mink disease parvovirus (ADV) is the etiological agent of Aleutian disease of mink. Several ADV isolates have been identified which vary in the severity of the disease they elicit. The isolate ADV-Utah replicates to high levels in mink, causing severe Aleutian disease that results in death within 6 to 8 weeks, but does not replicate in Crandell feline kidney (CrFK) cells. In contrast, ADV-G replicates in CrFK cells but does not replicate in mink. The ability of the virus to replicate in vivo is determined by virally encoded determinants contained within a defined region of the VP2 gene (M. E. Bloom, J. M. Fox, B. D. Berry, K. L. Oie, and J. B. Wolfinbarger. Virology 251:288-296, 1998). Within this region, ADV-G and ADV-Utah differ at only five amino acid residues. To determine which of these five amino acid residues comprise the in vivo replication determinant, site-directed mutagenesis was performed to individually convert the amino acid residues of ADV-G to those of ADV-Utah. A virus in which the ADV-G VP2 residue at 534, histidine (H), was converted to an aspartic acid (D) of ADV-Utah replicated in CrFK cells as efficiently as ADV-G. H534D also replicated in mink, causing transient viremia at 30 days postinfection and a strong antibody response. Animals infected with this virus developed diffuse hepatocellular microvesicular steatosis, an abnormal accumulation of intracellular fat, but did not develop classical Aleutian disease. Thus, the substitution of an aspartic acid at residue 534 for a histidine allowed replication of ADV-G in mink, but the ability to replicate was not sufficient to cause classical Aleutian disease.     

Cytokine profiles in adult mink infected with Aleutian mink disease parvovirus

The aim of this study was to examine the levels of gamma interferon (IFN-gamma)-, interleukin 4 (IL-4)-, and IL-8-producing cells in peripheral blood mononuclear cells from mink infected with the Aleutian mink disease parvovirus (ADV). As expected, ADV-infected mink developed high plasma gamma globulin values (hypergammaglobulinemia) and enhanced quantities of CD8-positive (CD8(+)) cells in the blood during the infection. We quantified the percentages of IFN-gamma- and IL-4-positive lymphocytes and IL-8-positive monocytes up to week 38 after virus challenge. The results clearly indicated marked increases in the percentages of IFN-gamma- and IL-4-producing lymphocytes during ADV infection. The total number of IL-8-producing monocytes in the blood of ADV-infected mink stayed fairly constant during the infection. In order to characterize the phenotype of the cytokine-producing cells, we performed double-labeling fluorescence-activated cell sorter (FACS) experiments with CD8 surface labeling in one channel and cytokine intracellular staining in the other. We found that most IFN-gamma and IL-4 in ADV-infected mink was produced by CD8(+) cells, while in the uninfected mink, these cytokines were primarily produced by a cell type that was not CD8 (possibly CD4-positive cells). We also observed that IL-8 was almost exclusively produced by monocytes. All of the above findings led us to conclude that both Th1- and Th2-driven immune functions are found in mink plasmacytosis.     

Immunoenzyme Western blotting analysis of antibody specificity in Aleutian disease of mink, a parvovirus infection.

Aleutian disease virus (ADV), an autonomous parvovirus, persistently infects mink and induces very high levels of virus-specific antibody. All strains of ADV infect all mink, but only highly virulent strains cause progressive disease in non-Aleutian mink. The development of antibody to individual ADV proteins was evaluated by Western blotting by using the sera of 22 uninfected mink and 163 naturally or experimentally infected mink. ADV has virion proteins of 86,000 and 78,000 daltons that are closely related. A new, possibly nonvirion protein of 143,000 daltons was observed, as well as a known nonvirion protein of 71,000 daltons. Sera from mink experimentally or naturally infected with ADV of high or low virulence generally reacted about equally with all four proteins. The only exceptions noted were that 8 of 15 sera of mink infected transplacentally preferentially reacted with the two virion proteins and sera from mink with the monoclonal gammopathy of Aleutian disease reacted preferentially with either virion (10 of 12) or nonvirion (2 of 12) proteins.     

Plateau levels of viremia correlate with the degree of CD4+-T-cell loss in simian immunodeficiency virus SIVagm-infected pigtailed macaques: variable pathogenicity of natural SIVagm isolates

Simian immunodeficiency virus from African green monkeys (SIVagm) results in asymptomatic infection in its natural host species. The virus is not inherently apathogenic, since infection of pigtailed (PT) macaques (Macaca nemestrina) with one isolate of SIVagm results in an immunodeficiency syndrome characterized by progressive CD4+-T-cell depletion and opportunistic infections. This virus was passaged once in a PT macaque and, thus, may not be entirely reflective of the virulence of the parental strain. The goal of the present study was to assess the pathogenicity of the PT-passaged isolate (SIVagm9063) and two primary SIVagm isolates in PT macaques, including the parental strain of the PT-passaged variant. Infection of macaques with any of the three isolates resulted in high levels of primary plasma viremia by 1 week after inoculation. Viremia was quickly controlled following infection with SIVagm155; these animals have maintained CD4+-T-cell subsets and remain healthy. The plateau levels among SIVagm90- and SIVagm9063-inoculated macaques varied widely from 100 to 1 million copies/ml of plasma. Three of four animals from each of these groups progressed to AIDS. Setpoint viremia and the degree of CD4+-T-cell loss at 6 months postinfection were not significantly different between macaques inoculated with SIVagm90 and SIVagm9063. However these parameters were significantly different in SIVagm155-inoculated macaques (P values of <0.01). Considering all the macaques, the degree of CD4+-T-cell loss by 6 months postinfection correlated with the plateau levels of viremia. Thus, similar to SIVsm/mac infection of macaques and human AIDS, viral load is an excellent prognostic indicator of disease course. The inherent pathogenicity of natural SIVagm isolates varies, but such natural isolates are capable of inducing AIDS in macaques without prior macaque passage.     

Activation of expression of two immunoglobulin SN-genes in the American mink with Aleutian disease

110 ranch-raised minks were injected with the Aleutian disease virus. Allotypes of constant regions of gamma-heavy chains of the mink immunoglobulins secreted have been analysed during 3 months. Activation of the expression of two markers (H3 and/or H4) up to minor or to nominal level (above 200 micrograms/ml) was observed. No such enhancement of expression of two other allotypes (H6 and H8) was found. The results suggest that the expression of two mink immunoglobulin CH genes induced by viral infection has allotype-specific regulation.     

The relationship between capsid protein (VP2) sequence and pathogenicity of Aleutian mink disease parvovirus (ADV): a possible role for raccoons in the transmission of ADV infections.

Aleutian mink disease parvovirus (ADV) DNA was identified by PCR in samples from mink and raccoons on commercial ranches during an outbreak of Aleutian disease (AD). Comparison of DNA sequences of the hypervariable portion of VP2, the major capsid protein of ADV, indicated that both mink and raccoons were infected by a new isolate of ADV, designated ADV-TR. Because the capsid proteins of other parvoviruses play a prominent role in the determination of viral pathogenicity and host range, we decided to examine the relationship between the capsid protein sequences and pathogenicity of ADV. Comparison of the ADV-TR hypervariable region sequence with sequences of other isolates of ADV revealed that ADV-TR was 94 to 100% related to the nonpathogenic type 1 ADV-G at both the DNA and amino acid levels but less than 90% related to other pathogenic ADVs like the type 2 ADV-Utah, the type 3 ADV-ZK8, or ADV-Pullman. This finding indicated that a virus with a type 1 hypervariable region could be pathogenic. To perform a more comprehensive analysis, the complete VP2 sequence of ADV-TR was obtained and compared with that of the 647-amino-acid VP2 of ADV-G and the corresponding VP2 sequences of the pathogenic ADV-Utah, ADV-Pullman, and ADV-ZK8. Although the hypervariable region amino acid sequence of ADV-TR was identical to that of ADV-G, there were 12 amino acid differences between ADV-G and ADV-TR. Each of these differences was at a position where other pathogenic isolates also differed from ADV-G. Thus, although ADV-TR had the hypervariable sequence of the nonpathogenic type 1 ADV-G, the remainder of the VP2 sequence resembled sequences of other pathogenic ADVs. Under experimental conditions, ADV-TR and ADV-Utah were highly pathogenic and induced typical AD in trios of both Aleutian and non-Aleutian mink, whereas ADV-Pullman was pathogenic only for Aleutian mink and ADV-G was noninfectious. Trios of raccoons experimentally inoculated with ADV-TR and ADV-Utah all became infected with ADV, but only a single ADV-Pullman-inoculated raccoon showed evidence of infection. Furthermore, none of the ADV isolates induced pathological findings of AD in raccoons. Finally, when a preparation of ADV-TR prepared from infected raccoon lymph nodes was inoculated into mink and raccoons, typical AD was induced in Aleutian and non-Aleutian mink, but raccoons failed to show serological or pathological evidence of infection. These results indicated that raccoons can become infected with ADV and may have a role in the transmission of virus to mink but that raccoon-to-raccoon transmission of ADV is unlikely.     

Antibodies to Aleutian mink disease parvovirus in free-ranging European mink (Mustela lutreola) and other small carnivores from southwestern France

Owing to the rapid decline of the European mink (Mustela lutreola) in France, a national conservation action plan has been initiated, in which scientific research to improve understanding of the causes of the decline is one of the primary objectives. In order to investigate the possible role of Aleutian disease parvovirus (ADV) in decline of the species, a serologic survey was conducted from March 1996 to March 2002 in 420 free-ranging individuals of six species of small carnivores distributed in eight departments of southwestern France. Antibodies to ADV were detected in 17 of 75 American mink (Mustela vison), 12 of 99 European mink, 16 of 145 polecats (Mustela putorius), four of 17 stone martens (Martes foina), one of 16 pine martens (Martes martes), and three of 68 common genets (Genetta genetta). Seroprevalence was significantly higher in American mink than in other species. Seropositive individuals with gamma globulin levels >20% were observed in four European mink, four American mink, two stone martens, and one pine marten. Geographic distribution of positive animals indicates the virus has spread to all areas where European mink are found. Furthermore, a trend of increasing prevalence seems to appear in Mustela sp. sympatric with American mink. Although further investigations are necessary to evaluate the role of ADV in decline of European mink, evidence of the virus in the wild at the levels found in our study has implications for conservation of this species.     

Inhibition of Adaptive Immune Responses Leads to a Fatal Clinical Outcome in SIV-Infected Pigtailed Macaques but Not Vervet African Green Monkeys

African green monkeys (AGM) and other natural hosts for simian immunodeficiency virus (SIV) do not develop an AIDS-like disease following SIV infection. To evaluate differences in the role of SIV-specific adaptive immune responses between natural and nonnatural hosts, we used SIV_agmVer90 to infect vervet AGM and pigtailed macaques (PTM). This infection results in robust viral replication in both vervet AGM and pigtailed macaques (PTM) but only induces AIDS in the latter species. We delayed the development of adaptive immune responses through combined administration of anti-CD8 and anti-CD20 lymphocyte-depleting antibodies during primary infection of PTM (n = 4) and AGM (n = 4), and compared these animals to historical controls infected with the same virus. Lymphocyte depletion resulted in a 1-log increase in primary viremia and a 4-log increase in post-acute viremia in PTM. Three of the four PTM had to be euthanized within 6 weeks of inoculation due to massive CMV reactivation and disease. In contrast, all four lymphocyte-depleted AGM remained healthy. The lymphocyte-depleted AGM showed only a trend toward a prolongation in peak viremia but the groups were indistinguishable during chronic infection. These data show that adaptive immune responses are critical for controlling disease progression in pathogenic SIV infection in PTM. However, the maintenance of a disease-free course of SIV infection in AGM likely depends on a number of mechanisms including non-adaptive immune mechanisms.     

High prevalence of Aleutian mink disease virus in free-ranging mink on a remote Danish island

Aleutian mink disease virus (AMDV) causes severe disease in farmed mink (Neovison vison) worldwide. In Denmark, AMDV in farmed mink has been confined to the northern part of the mainland since 2002. From 1998 to 2009, samples from 396 free-ranging mink were collected from mainland Denmark, and a low AMDV antibody prevalence (3% of 296) was found using countercurrent immune electrophoresis. However, on the island of Bornholm in the Baltic Sea, a high prevalence (45% of 142 mink) was detected in the free-ranging mink. Aleutian mink disease virus was detected by polymerase chain reaction in 32 of 49 antibody-positive free-ranging mink on Bornholm, but not in mink collected from other parts of Denmark. Sequence analysis of 370 base pairs of the nonstructural gene of the AMDV of 17 samples revealed two clusters with closest similarity to Swedish AMDV strains.     

Studies on the Progression of Aleutian Disease in Mink

One hundred ninetyfive male ADV-negative mink, including 79 pairs of brothers, were followed in their response to natural ADV-infection caused by mating with ADV-positive females and under epidemic conditions. Special attention was drawn to the development of progressive versus non-progressive Aleutian disease. This was done by plasmaelectrophoresis, detection of antibodies to ADV, and finally by macroscopical examination of mink organs at pelting time. We found that the progression of Aleutian disease presumably is under some genetic influence. We also found indication of differences in the response to ADV depending on how the infection was introduced. Mating to positive females (low virus concentration) resulted in significantly higher proportion of non-progressive responders than infection under epidemic conditions (high virus concentration).     

Identification of a nonvirion protein of Aleutian disease virus: mink with Aleutian disease have antibody to both virion and nonvirion proteins.

We studied Aleutian disease virus polypeptides in Crandall feline kidney (CRFK) cells. When CRFK cells labeled with [35S]methionine at 60 h postinfection were studied by immunoprecipitation with sera from infected mink, the major Aleutian disease virus virion polypeptides (p85 and p75) were consistently identified, as was a 71,000-dalton nonvirion protein (p71). The peptide maps of p85 and p75 were similar, but the map of p71 was different. p85, p75, and p71 were all precipitated by sera from Aleutian disease virus-infected mink, including those with signs of progressive disease, but heterologous sera raised against purified Aleutian disease virus did not precipitate the nonvirion p71. These results indicated that the nonvirion p71 was unrelated to p85 and p75 and further suggested that mink infected with Aleutian disease virus develop antibody to nonvirion, as well as structural, viral proteins.     

Replication of Aleutian mink disease parvovirus in lymphoid tissues of adult mink: involvement of follicular dendritic cells and macrophages.

By using strand-specific in situ hybridization and immunohistochemistry, evidence for replication of the Aleutian mink disease parvovirus was observed in cells resembling macrophages and cells resembling follicular dendritic cells at 10 days after infection but only in macrophages at 60 days. Sequestration of the Aleutian mink disease parvovirus in larger numbers of macrophages and follicular dendritic cells was noted at both 10 and 60 days.     

Experimental studies on vertical infection of mice with Japanese encephalitis virus. IV. Effect of virus strain on placental and fetal infection

An experiment was carried out to examine the effect of an inoculated strain of Japanese encephalitis virus on the establishment of experimental vertical infection of mice with this virus. In it, closed-colony mice of the CFW strain were inoculated intravenously with seven strains of the virus at 7 days of pregnancy. After that, an attempt was made to recover the virus from placenta and fetus, so that the infection rate of each strain might be determined. As a result, the infection rate was high for both placenta and fetus in the case of the AS-6 and Sagara strains both of which had undergone three passages in the mouse brain. The placental infection rate was high and the fetal infection rate relatively low in the case of the JaGAr01 and Fuji strains which had undergone 7 and 150 passages, respectively, in the mouse brain. The infection rate was very low for both placenta and fetus in the case of the Nakayama-Yakken strain which had undergone more than 100 passages in the mouse brain. There was no difference in the severity of viremia after inoculation between the AS-6 and Fuji strains. Both placental and fetal infection rates were low in the case of the JaTH160 strain which had undergone passages in mice by intraperitoneal inoculation and which presented a strong peripheral infectivity and induced a severe viremia after inoculation. Neither placental nor fetal infection occurred in the case of the S- strain used as live virus vaccine. These results indicated that placental and fetal infection rates varied from one virus strain to another.(ABSTRACT TRUNCATED AT 250 WORDS)     

Viral factors determine progression to AIDS in simian immunodeficiency virus-infected newborn rhesus macaques.

To evaluate how viral variants may affect disease progression in human pediatric AIDS, we studied the potential of three simian immunodeficiency virus (SIV) isolates to induce simian AIDS in newborn rhesus macaques. The three virus isolates were previously shown to range from pathogenic (SIVmac251 and SIVmac239) to nonpathogenic (SIVmac1A11) when inoculated intravenously into juvenile and adult rhesus macaques. Six newborn macaques inoculated with pathogenic, uncloned SIVmac251 developed persistent, high levels of cell-associated and cell-free viremia, had no detectable antiviral antibodies, and had poor weight gain; these animals all exhibited severe clinical disease and pathologic lesions diagnostic for simian AIDS and were euthanatized 10 to 26 weeks after inoculation. Two newborns inoculated with pathogenic, molecularly cloned SIVmac239 developed persistent high virus load in peripheral blood, but both animals had normal weight gain and developed antiviral antibodies. One of the SIVmac239-infected neonates exhibited pathologic lesions diagnostic for SAIDS and was euthanatized at 34 weeks after inoculation; the other SIVmac239-infected neonate remained alive and exhibited no significant clinical disease for more than 1 year after inoculation. In contrast, three newborn rhesus macaques inoculated with the nonpathogenic molecular clone, SIVmac1A11, had transient, low-level viremia, seroconverted by 10 weeks after inoculation, had normal weight gain, and remained healthy for over 1 year. These results indicate that (i) newborn rhesus macaques infected with an uncloned, virulent SIVmac isolate have a more rapid, fulminant disease course than do adults inoculated with the same virus, (ii) the most rapid disease progression is associated with lack of a detectable humoral immune response in SIV-infected infant macaques, (iii) a molecularly cloned, attenuated SIV isolate is nonpathogenic in neonatal macaques, and (iv) SIV-infected neonatal macaques exhibit patterns of infection, virus load, and disease progression similar to those observed in human immunodeficiency virus-infected children. This SIV/neonatal rhesus model of pediatric AIDS provides a rapid, sensitive model with which to compare the virulence of SIV isolates and to study the mechanisms underlying the differences in disease progression in human immunodeficiency virus-infected infants.