Magnetic protein microbead-aided indirect fluoroimmunoassay for the determination of canine virus specific antibodies

Rabies, canine distemper, and canine parvovirus are common contagious viral diseases of dogs and many other carnivores, and pose a severe threat to the population dynamics of wild carnivores, as well as endangering carnivore conservation. However, clinical diagnosis of these diseases, especially canine distemper and canine parvovirus, is difficult because of the broad spectrum of symptoms that may be confused with other respiratory and enteric diseases of dogs. The most frequently used and proven techniques for diagnosing viral diseases include the conventional enzyme-linked immunosorbent assay (ELISA), rapid fluorescent focus inhibition test (RFFIT), mouse neutralisation test (MNT), and fluorescent antibody virus neutralization (FAVN) test. However, these methods still have some inherent limitations. In this study, a magnetic protein microbead-aided indirect fluoroimmunoassay was developed to detect canine virus specific antibodies, human rabies immunoglobulin, CDV McAbs, and CPV McAbs. In this assay, an avidin-biotin system was employed to combine magnetic microbeads and virus antigens (rabies virus, canine distemper virus, and canine parvovirus). Quantification of the targeted virus antibodies was analyzed through indirect fluoroimmunoassay using the specific antigen-antibody reaction, as well as their corresponding FITC-labeled detection antibodies (mouse anti-human IgG/FITC conjugate or rabbit anti-dog IgG/FITC conjugate). The results indicated that the fluorescence intensity increased when a higher concentration of the targeted analyte was used, but the control had almost no fluorescence, much like the conventional ELISA. For human rabies immunoglobulin, CDV McAbs, and CPV McAbs, the minimum detectable concentrations were 0.2 IU/mL, 0.3 ng/mL, and 0.5 ng/mL, respectively. All of these results indicate that this assay can be employed to determine the presence of canine virus specific antibodies. In addition, the method devised here can be utilized as a general protocol in other bacterial and viral marker analysis.     

Immunological effects of recombinant feline interferon-omega (KT-80) administration in the dog

The immunological effects of recombinant feline interferon-omega (rFeIFN-omega ; KT-80, Toray) were examined on administration to healthy dogs. The activities of whole blood cells, macrophages, and natural killer cells were enhanced. Moreover, the whole blood activity was examined when KT-80 was administered to dogs which had been diagnosed as having natural canine parvovirus (CPV) infection. Only some cases in which the activity increased until 3 hr post-administration survived. These results suggest that rFeIFN-omega (KT-80) treatment enhanced the cellular immunity of normal dogs, and could exert significant therapeutic effects on only natural CPV infected dogs with induced continuous immunoenhancement.     

In situ hybridization for the detection of human parvovirus B19 nucleic acid sequences in paraffin-embedded specimens

Parvovirus infection of pregnant women leading to a transplacentar infection of the fetus may result in hydrops fetalis, and ultimately in intrauterine death of the fetus. In situ hybridization with a biotinylated as well as with a 35S-labeled probe for human parvovirus B19 was performed on formalin-fixed paraffin-embedded (FFPE) tissue from a fetus suffering from non-immunologic hydrops fetalis. Histology was suggestive of viral infection probably with human parvovirus. Parvovirus DNA could be detected and precisely localized mainly in the nuclei of erythroid precursors cells within fetal blood vessels of all organs examined. There was no detection of B19 nucleic acid in parenchymal cells of the placenta or the fetal organs, nor within maternal blood cells. These findings are in agreement with the well-known properties of animal parvoviruses to replicate exclusively in proliferating cells. Taking into consideration the problems in diagnosing human parvovirus infection by light microscopy, we conclude that in situ hybridization with an appropriate non-radioactive probe is a valuable, rapid and safe complementary detection method for the diagnosis and study of human parvovirus infections. The 35S-labeled probe is more sensitive than the biotinylated probe, but has the disadvantages of lower resolution of the signal, longer duration of the assay, the hazard of radioactivity and the shorter shelf-life of the probe.     

Evolution of the feline-subgroup parvoviruses and the control of canine host range in vivo.

A related group of parvoviruses infects members of many different carnivore families. Some of those viruses differ in host range or antigenic properties, but the true relationships are poorly understood. We examined 24 VP1/VP2 and 8 NS1 gene sequences from various parvovirus isolates to determine the phylogenetic relationships between viruses isolated from cats, dogs, Asiatic raccoon dogs, mink, raccoons, and foxes. There were about 1.3% pairwise sequence differences between the VP1/VP2 genes of viruses collected up to four decades apart. Viruses from cats, mink, foxes, and raccoons were not distinguished from each other phylogenetically, but the canine or Asiatic raccoon dog isolates formed a distinct clade. Characteristic antigenic, tissue culture host range, and other properties of the canine isolates have previously been shown to be determined by differences in the VP1/VP2 gene, and we show here that there are at least 10 nucleotide sequence differences which distinguish all canine isolates from any other virus. The VP1/VP2 gene sequences grouped roughly according to the time of virus isolation, and there were similar rates of sequence divergence among the canine isolates and those from the other species. A smaller number of differences were present in the NS1 gene sequences, but a similar phylogeny was revealed. Inoculation of mutants of a feline virus isolate into dogs showed that three or four CPV-specific differences in the VP1/VP2 gene controlled the in vivo canine host range.     

In situ hybridization for the detection of human parvovirus B19 nucleic acid sequences in paraffin-embedded specimens

Parvovirus infection of pregnant women leading to a transplacentar infection of the fetus may result in hydrops fetalis, and ultimately in intrauterine death of the fetus. In situ hybridization with a biotinylated as well as with a³⁵S-labeled probe for human parvovirus B19 was performed on formalin-fixed paraffin-embedded (FFPE) tissue from a fetus suffering from non-immunologic hydrops fetalis. Histology was suggestive of viral infection probably with human parvovirus. Parvovirus DNA could be detected and precisely localized mainly in the nuclei of erythroid precursors cells within fetal blood vessels of all organs examined. There was no detection of B19 nucleic acid in parenchymal cells of the placenta or the fetal organs, nor within maternal blood cells. These findings are in agreement with the well-known properties of animal parvoviruses to replicate exclusively in proliferating cells. Taking into consideration the problems in diagnosing human parvovirus infection by light microscopy, we conclude that in situ hybridization with an appropriate non-radioactive probe is a valuable, rapid and safe complementary detection method for the diagnosis and study of human parvovirus infections. The³⁵S-labeled probe is more sensitive than the biotinylated probe, but has the disadvantages of lower resolution of the signal, longer duration of the assay, the hazard of radioactivity and the shorter shelflife of the probe.     

High viral load of human bocavirus correlates with duration of wheezing in children with severe lower respiratory tract infection

Background

Human bocavirus (HBoV) is a newly discovered parvovirus and increasing evidences are available to support its role as an etiologic agent in lower respiratory tract infection (LRTI). The objective of this study is to assess the impact of HBoV viral load on clinical characteristics in children who were HBoV positive and suffered severe LRTI.

Methods

Lower respiratory tract aspirates from 186 hospitalized children with severe LRTI were obtained by bronchoscopy. HBoVs were detected by real-time PCR and other 10 infectious agents were examined using PCR and/or direct fluorescent assay.

Results

Thirty-one patients (24.6%) were tested positive for HBoV in the respiratory tract aspirates. Fifteen samples had a high viral load (>10⁴ copies/mL) and the other sixteen samples had a low viral load (<10⁴ copies/mL). The duration of presented wheezing and hospitalization was longer in children with high viral load of HBoV than that in children with low viral load. The days of wheezing showed a correlation with viral load of HBoV.

Conclusion

We confirmed that HBoV was frequently detected in patients with severe LRTI. Wheezing was one of the most common symptoms presented by patients with positive HBoV. A high HBoV viral load could be an etiologic agent for LRTI, which led to more severe lower respiratory tract symptom, longer duration of wheezing and hospitalization.     

Maternally-derived antibodies in pups and protection from canine parvovirus infection

The interaction between maternally-derived antibodies (MDA) and canine parvovirus (CPV) infection was evaluated in five groups of pups with a wide range of haemagglutination inhibiting (HI) titres of MDA (from < 10 to 320). The pups were inoculated with a field CPV strain and monitored daily to evaluate their clinical condition and viral shedding in the faeces. Serum samples were collected weekly to evaluate antibody response. Clinical signs were observed in dogs with HI titres up to 80. Active CPV replication was demonstrated in dogs with HI titres up to 160, although slightly delayed, at lower titres and for a shorter period compared to seronegative dogs. The successful infection of dogs with HI titres of 80 and 160 was also confirmed by seroconversion, evaluated at day 14 postinfection. These findings are in contrast with the MDA titre (HI > or = 80) usually considered fully protective for CPV infection, and suggest the need for revision of current vaccination programmes for pups.     

Evolutionary Reconstructions of the Transferrin Receptor of Caniforms Supports Canine Parvovirus Being a Re-emerged and Not a Novel Pathogen in Dogs

Parvoviruses exploit transferrin receptor type-1 (TfR) for cellular entry in carnivores, and specific interactions are key to control of host range. We show that several key mutations acquired by TfR during the evolution of Caniforms (dogs and related species) modified the interactions with parvovirus capsids by reducing the level of binding. These data, along with signatures of positive selection in the TFRC gene, are consistent with an evolutionary arms race between the TfR of the Caniform clade and parvoviruses. As well as the modifications of amino acid sequence which modify binding, we found that a glycosylation site mutation in the TfR of dogs which provided resistance to the carnivore parvoviruses which were in circulation prior to about 1975 predates the speciation of coyotes and dogs. Because the closely-related black-backed jackal has a TfR similar to their common ancestor and lacks the glycosylation site, reconstructing this mutation into the jackal TfR shows the potency of that site in blocking binding and infection and explains the resistance of dogs until recent times. This alters our understanding of this well-known example of viral emergence by indicating that canine parvovirus emergence likely resulted from the re-adaptation of a parvovirus to the resistant receptor of a former host.     

Parvovirus host range,cell tropism and evolution

The past few years have seen major advances in our understanding of the controls of evolution, host range and cell tropism of parvoviruses. Notable findings have included the identification of the transferrin receptor TfR as the cell surface receptor for canine parvovirus and feline panleukopenia virus, and also the finding that specific binding to the canine TfR led to the emergence of canine parvovirus as a new pathogen in dogs. The structures of the adeno-associated virus-2 and porcine parvovirus capsids, along with those of the minute virus of mice, have also advanced our understanding of parvovirus biology. Structure-function studies have shown that in several different parvoviruses the threefold spikes or peaks of the capsid control several aspects of cell tropism and host range, and that those are subject to selective pressures leading to viral evolution. The cell and tissue tropisms of different adeno-associated virus serotypes were demonstrated to be due, in part, to specific receptor binding.     

First peptide vaccine providing protection against viral infection in the target animal: studies of canine parvovirus in dogs.

A synthetic peptide vaccine which protects dogs against challenge with virulent canine parvovirus is described. The amino acid sequence used was discovered in previous studies on the immunogenic properties of previously mapped antigenic sites and represents the amino-terminal region of viral protein VP2. As with marker vaccines, it is possible to discriminate between vaccinated dogs that have not been exposed to the virus and dogs that have been infected with the virus. The protective mechanism can be explained by a humoral response against the peptide aided by T-cell epitopes contained in the carrier protein used for peptide coupling. This is the first example of a synthetic peptide vaccine that induces protection in target animals.     

Replication of B19 parvovirus in highly enriched hematopoietic progenitor cells from normal human bone marrow.

The target cell specificity of the B19 parvovirus infection was examined by isolating highly enriched hematopoietic progenitor and stem cells from normal human bone marrow. The efficiency of the B19 parvovirus replication in enriched erythroid progenitor cells was approximately 100-fold greater than that in unseparated bone marrow cells. The more-primitive progenitor cells identical to or closely related to the human pluripotent hematopoietic stem cells, on the other hand, did not support viral replication. The B19 progeny virus produced by the enriched erythroid progenitor cells was infectious and strongly suppressed erythropoiesis in vitro. The susceptibility of both the more-primitive erythroid progenitors (burst-forming units-erythroid) and the more-mature erythroid progenitors (CFU-erythroid) to the cytolytic response of the virus and the lack of effect on the myeloid progenitors (CFU-granulocyte-macrophage) further give evidence to the remarkable tropism of the B19 parvovirus for human hematopoietic cells of erythroid lineage.     

Studies of respiratory disease in random-source laboratory dogs: viral infections in unconditioned dogs.

A prospective study was conducted to identify the viruses causing respiratory diseases in unconditioned, random-source dogs. During the quarantine period, respiratory disease occurred in 86 of 167 (52%) dogs, and 34 (21%) died. Most affected dogs had a distemper-like illness which required extensive and prolonged care. Histopathologic studies confirmed the diagnosis of canine distemper in 10 of 12 (83%) fatal infections examined. Sixty-seven of 91 (74%) dogs which arrived without canine distemper antibody became ill, and 30 (32%) died. In contrast, only 16 of 67 (24%) dogs with canine distemper antibody had respiratory disease, and only 3 (4%) died. Parainfluenza SV5 and canine adenovirus--type II were recovered from 27 of 54 and 22 of 54 sick dogs, respectively. Canine herpesviruses, canine coronaviruses, and canine parvoviruses were less frequently isolated. Increased antibody titers to SV5 were found consistently, and rises in titer to the other viruses were demonstrated. Many of the sick dogs were infected with two or more viruses. Although several viral agents were detected during these epizootics, prevention of canine distemper appeared to be the key to controlling severe, prolonged, and often fatal respiratory disease.     

Immunogenicity of the capsid protein VP2 from porcine parvovirus expressed in low alkaloid transgenic tobacco

Porcine parvovirus is a widespread infectious viral disease with serious consequences to the reproductive health of swine. We have expressed the VP2 capsid protein of porcine parvovirus in the leaves of low alkaloid transgenic tobacco at approximately 0.3% of total soluble protein. Self-assembled virus-like particles were observed in planta by electron microscopy. Total soluble protein was extracted from the plant tissue and administered to mice by subcutaneous injection. An immune response was detected in these mice. The ability of serum antibodies to neutralize the infectivity of porcine parvovirus was further examined by a serum neutralization assay and was determined to be 1:2700–1:3900, a clear indication of the potential of VP2 expressed in plant material as a subunit vaccine against porcine parvovirus.     

Role of multiple hosts in the cross-species transmission and emergence of a pandemic parvovirus

Understanding the mechanisms of cross-species virus transmission is critical to anticipating emerging infectious diseases. Canine parvovirus type 2 (CPV-2) emerged as a variant of a feline parvovirus when it acquired mutations that allowed binding to the canine transferrin receptor type 1 (TfR). However, CPV-2 was soon replaced by a variant virus (CPV-2a) that differed in antigenicity and receptor binding. Here we show that the emergence of CPV involved an additional host range variant virus that has circulated undetected in raccoons for at least 24 years, with transfers to and from dogs. Raccoon virus capsids showed little binding to the canine TfR, showed little infection of canine cells, and had altered antigenic structures. Remarkably, in capsid protein (VP2) phylogenies, most raccoon viruses fell as evolutionary intermediates between the CPV-2 and CPV-2a strains, suggesting that passage through raccoons assisted in the evolution of CPV-2a. This highlights the potential role of alternative hosts in viral emergence.     

Studies on the sequential development of acute interstitial pneumonia caused by Aleutian disease virus in mink kits.

We studied different parameters during the development of acute interstitial pneumonia in mink kits caused by neonatal infection with Aleutian disease virus (ADV). When histological lesions, presence of intranuclear inclusion bodies, and intranuclearly localized ADV antigen were correlated with levels of single-stranded virion and duplex replicative forms of ADV DNA in the different tissues, it was concluded that the lung, probably alveolar type II cells, is the major primary target for viral replication and cytopathology. The presence of the duplex dimeric replicative-form DNA, a strong marker of parvovirus replication, was also observed in low amount in the mesenteric lymph node, suggesting replication of ADV in this organ, although no viral cytopathology could be demonstrated. Moreover, a few intranuclear inclusion bodies were demonstrated in kidney and liver from affected kits, but intranuclearly localized ADV antigen could not be demonstrated in liver sections, and neither could duplex dimer replicative-form DNA, suggesting that these organs are nevertheless not a major site of ADV replication. When the data were compared with results previously reported for ADV-infected adult mink and ADV-infected permissive cell cultures, the data suggested that the pattern of ADV replication in alveolar type II cells is similar to that seen in infected cell cultures but that the replication in the other kit organs resembles the restricted pattern seen in adult mink.     

Possible "immuno-protection" of the bovine parvovirus in the uterus: Preliminary communication

A pregnant Holstein cow was experimentally inoculated with bovine parvovirus. Approximately five weeks after the last of three injections, the animal aborted a fetus which showed signs of mummification. Virus particles similar to bovine parvovirus were identified by electron microscopy from fetal lung tissue and intestinal contents. Five months after the abortion, the experimental animal died of a corynebacterial metritis and septicemia. Immunofluorescence tests were performed on representative tissues obtained at necropsy. Of all tissues examined only the uterus showed a positive response to the immunofluorescence assay, indicating that the viral antigen persisted in the uterine environment long after its elimination from the systemic circulation.     

Replication Process of the Parvovirus H-1 I. Kinetics in a Parasynchronous Cell System

Parasynchronous cultures of hamster embryo cells were used to study some of the events in the replication process of the parvovirus H-1. Synthesis of viral DNA, viral hemagglutinating antigen, and infectious virus were examined. It was found that initiation of DNA synthesis, on which subsequent viral hemagglutinin synthesis was dependent, occurred at a specific time in late S-phase. Production of H-1 viral protein was shown to be sensitive to inhibition by alpha-amanitin. The implications of these findings are discussed.     

Characterization of mouse parvovirus infection by in situ hybridization.

Infection of young adult BALB/cByJ mice with mouse parvovirus-1, a newly recognized, lymphocytotropic, nonpathogenic parvovirus, was examined by in situ hybridization. Virus appeared to enter through the small intestine and was disseminated to the liver and lymphoid tissues. Strand-specific probes detected virion DNA in a consistently larger number of cells than replicative forms of viral DNA and/or viral mRNA. The number of signal-positive cells in the intestinal mucosa, lymph nodes, spleen, and thymus increased through day 10 after oral inoculation but decreased after seroconversion. Positive cells were still detected, however, in peripheral lymphoid tissues of mice examined at 9 weeks postinoculation. The results underscore the need to assess potential effects of persistent mouse parvovirus-1 infection on immune function in mice.     

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