key: cord-0010003-eofm2lqv
authors: Osterhaus, A. D. M. E.; van Steenis, G.; de Kreek, P.
title: Isolation of a Virus Closely Related to Feline Panleukopenia Virus from Dogs with Diarrhea
date: 2010-05-13
journal: J Vet Med B Infect Dis Vet Public Health
DOI: 10.1111/j.1439-0450.1980.tb01632.x
sha: 22b1c0eba01daf331d75c0ae4646386a446ab49b
doc_id: 10003
cord_uid: eofm2lqv

SUMMARY: Parvovirus‐like particles were demonstrated by negative contrast electron microscopy in feces of dogs involved in outbreaks of a contagious diarrhea in the Netherlands. The virus was isolated in a continuous cell line in which it produced intranuclear inclusion bodies. It showed a close antigenic relationship with feline panleukopenia virus: a two‐way cross‐reaction was observed in hemagglutination inhibition, immune electron microscopy, immunofluorescence and serum neutralization tests. ZUSAMMENFASSUNG: Isolierung eines mit dem Virus der felinen Panleukopenie verwandten Virus von Hunden mit Diarrhoe Parvovirus‐ähnliche Partikel wurden mit Hilfe der Negativkontrast‐Elektronenmikroskopie während eines Ausbruches von kontagiöser Diarrhoe in Fäces von Hunden in den Niederlanden nachgewiesen. Das Virus wurde in einer permanenten Zell‐Linie isoliert, in der intranukleäre Einschlußkörperchen nachgewiesen werden konnten. Es zeigte eine nahe Antigenverwandt‐schaft mit dem Virus der felinen Panleukopenie. Eine beiderseitige Kreuzreaktion wurde beobachtet in der Hämagglutinationshemmung, Immunelektronenmikroskopie, Immunofluoreszenz und im Serumneutralisations‐Tests. RÉSUMÉ: Isolement d'un virus très proche au virus de la panleucopénie féline chez des chiens diarhéiques Des particules ressemblant aux parvovirions ont été démontrées au microscope électronique (contraste‐négatif) dans les matières fécales des chiens diarrhéiques aux Pays‐Bas. Le virus était isolé sur une ligne cellulaire continue dans laquelle il produisait des corpuscules d'inclusion intranucléairs. Le virus manifestait un parenté antigénique avec le virus de la panleucopénie féline: des réactions croisées dans les deux sens étaient observées, utilisant des méthodes d'inhibition d'hémagglutination, immunomicroscopie électronique, immunofluorescence et séroneutralisation. RESUMEN: Aislamiento de un virus estrechamente relacionado con el virus de la panleucopenia felina en perros con diarrea Partículas semejantes a parvoviriones fueron demostrados por medio de microscopía electrónica (contraste negativo) en muestras fecales de perros durante endemias de diarrea infecciosa en los Paises Bajos. Se mostró el aislamiento del virus en una línea celular continua en donde producía corpúsculos de inclusión intranucleares. El virus tiene una relación antigénica estrecha con el virus de la panleucopenia felina: reacciones cruzadas en ambos sentidos fueron observadas por medio de pruebas de inhibición de la hemaglutinación, inmunomicroscopía electrónica, inmunofluorescencia y sueroneutralización.

The first isolation of a parvovirus from dogs, the Minute Virus of Canines (MVC) dates from 1970 (3). The virus which was isolated from stools of healthy dogs did not appear to be associated with disease symptoms and no antigenic relationship with any of the known parvoviruses including feline panleukopenia virus (FPV) could be demonstrated (3, 15) .

During the last two years parvovirus-like particles were shown to be present in feces of dogs involved in several outbreaks of a contagious diarrhea often accompanied with vomiting, in the USA, Canada and Australia (1, 4, 5, 6, 8, 10) . In many cases intestinal lesions were very similar to those of feline panleukopenia (1, 4, lo) . A virus isolated from dogs in these outbreaks was shown to be antigenically related to FPV on the basis of a cross-reactivity in immunofluorescence (IF) tests (2,4) and in serum neutralization tests (4).

In the present paper we describe the demonstration of parvovirus-like particles in stools of dogs in outbreaks of contagious diarrhea often associated with vomiting in the Netherlands. Mortality rates were high, especially among dogs under one year of age. Intestinal lesions very similar to those of feline panleukopenia were often observed in these dogs (7) . The virus isolated from stools of an affected dog showed a close antigenic relationship with FPV. 12 OSTERHAUS, VAN STEENIS and DE KREEK Zeist, The Netherlands). N o indications of the presence of known feline viruses have ever been found in regular screening procedures in this surgically derived colony (OSTERHAUS, unpublished observations). The animals were kept in a pressurized glove box throughout the experiments.

Two non-vaccinated conventionally reared male dogs about four months old were also made available by Dr. A. F. ANGULO. They were kept in an isolated room throughout the experiments.

A vaccine strain of FPV ("Dohyvac P") together with the Crandell Feline Kidney (CrFK) cell line were kindly provided by Dr. C. FOLKERS (Philips-Duphar, Weesp, The Netherlands). The virus was propagated in monolayer cultures of these cells in Leighton tubes or in Roux bottles by adding the virus suspensions to cell suspensions or to settled monolayers respectively. The medium used was similar to the medium used for virus isolation procedures (see below). After an incubation period of two to three days at 37 "C, the Leighton tube cultures were used for indirect IF studies and the bottle cultures were centrifuged at low speed after three cycles of freezing and thawing; the supernatants were used for hemagglutination (HA), (immune) electron microscopy (IEM) and serum neutralization (SN) studies.

Stools were taken from dogs involved in the outbreaks of diarrhea in the Netherlands either in a breeding colony for experimental animals, or in boarding kennels. Serum samples were collected from dogs in the breeding colony after the outbreaks had started. In addition sera were tested from dogs of the breeding colony and from the open population in the Netherlands, which had been collected about four years ago (11) .

Fecal samples were homogenized in 10 volumes of PBS and centrifuged a t 10,000 g. for 5 minutes. Material from the supernatant-pellet interface was used as a fecal extract for EM and IEM.

For negative contrast EM the fecal extracts were applied to carbon-coated grids. After 10 minutes the grids were rinsed with distilled water and contrasted with a solution of 2 O/o phosphotungstic acid, containing 0.01 O/o bacitracin and adjusted to p H 6.2 with KOH.

For IEM the samples were mixed with equal volumes of 1 : 10 diluted immune sera or control sera and incubated for two hours at 37OC. The reactant mixtures were applied to carbon-coated grids and negatively contrasted as described above. Grids were examined at a magnification of 50,000 using a JEOL 100 C electron microscope.

Fecal samples were diluted 1 : 10 in PBS and shaken for 15 minutes with chloroform at 4 OC. After low speed centrifugation 0.15 ml. volumes of the aqueous phases were mixed in triplicate with 1.5 ml. volumes of a freshly trypsinized suspension of Madin Darby Canine Kidney (MDCK) cells. The cells were permitted to settle and grow on cover slips in Leighton tubes. The suspension contained about 2 x 106 cells per ml. in Eagles MEM-lactalbumin hydrolysate, supplemented with 10 Vo fetal calf serum and antibiotics. After an incubation period of two or three days the cultures were examined for cytopathic changes. One tube per sample was used for haematoxylin and eosin (HE) staining (standard procedure), one for possible demonstration of viral antigen in an indirect IF test and one for subsequent passaging. For reisolation the procedure used was similar to the original isolation procedure. However, the material was passed through a 220 nm filter (Millipore@) after treatment with chloroform.

H A and HA1 tests were carried out in a microtiter system (Cooke Engineering Co.) essentially as described previously (1 1) using veronal gelatin buffer containing 0.15 O/o bovine serum albumin as a clikent. Hemagglutinating antigen was prepared from a canine parvovirus-like virus (CPV), isolated during the course of these studies, by centrifuging infected Roux bottle cultures three days post-infection (p. i.) at low speed after three cycles 13 of freezing and thawing. The supernatant was used as CPV hemagglutinating antigen. Likewise, the supernatant from FPV infected CrFK cell cultures treated in the same way were used as antigen for HA and HA1 tests. HA tests were carried out by adding a 0.75 O/o suspension from African green monkey (AGM), dog, cat, swine, mouse, rat, guinea pig, Chinese hamster or chicken erythrocytes, to serial twofold dilutions of the antigea preparations. After an incubation time of two hours at 4 O C the tests could be read. For HA1 tests serial twofold dilutions of kaolin treated (14) antisera were mixed with four hemagglutinating units (HAU). After an incubation period of one hour at 20 OC, a 0.75 O/o erythrocyte suspension was added and the tests were further incubated and read as the HA tests. Titers < 20 were regarded as non-specific.

Two or three days p.i. indirect IF tests were performed on Leighton tube cultures of MDCK or CrFK cultures after cold (-70 "C) ethanol fixation, to demonstrate CPV or FPV antigen. The tests were essentially carried out as described previously for a coronavirus system (12) . A rabbit anti-dog IgG and a rabbit anti-cat IgG preparation kindly provided by Dr. J. NAGEL (Natl. Inst. Publ. Hlth.), both labelled with FITC, were used as conjugates. When canine sera were used on MDCK cells or when feline sera were used on CrFK cells a FITC labelled protein-A preparation, also provided by Dr. J. NAGEL, was used instead of the anti-IgG conjugates.

Neutralization of CPV or FPV with inactivated (30 min 56 "C) sera was demonstrated by determining the neutralization indices in MDCK or CrFK Leighton tube cultures respectively: Semi log 10 dilutions of the virus suspensions were mixed with equal volumes of 1 : 25 diluted sera and incubated for one hour at 20 OC. Volumes of 0.5 ml. of these mixtures were added simultaneously to Leighton tube cultures with MDCK or CrFK cell suspensions. Three tubes were inoculated with each virus-serum mixture. After two or three days the cultures were fixed in 40/0 formaldehyde and stained with HE and examined for the presence of intranuclear inclusion bodies. From these results neutralization indices were calculated.

After collecting preinoculation serum samples of the two cats, one was vaccinated with two doses of a formaldehyde-inactivated panleukopenia vaccine ("Felidovac" Behringwerke AG, Marburg, GDR). After four weeks this cat was boosted with another two doses of the same vaccine and serum samples were collected from both cats one week later (post-vaccination and second serum sample respectively). Two weeks after the second vaccination, both cats were challenged with CPV, isolated during the course of these experiments: 1 ml. of a CPV suspension, containing about 104 TCID,, was inoculated intranasally and 1 ml. subcutaneously in both cats. The cats were checked for clinical signs daily, which included measuring of rectal temperature. White blood cell (WBC) counts were made every second day.

After collecting pre-inoculation serum samples, the dogs were infected by application of about 104 TCID,, CPV directly into their stomachs using an oesophageal tube. The dogs were checked daily for overt clinical signs. Post-infection sera were collected from the dogs 2ldays after experimental infection.

Stools from eight dogs in the breeding colony and from eight dogs in boarding kennels, which all died with symptoms of acute diarrhea, were examined in negative contrast electron microscopy. In seven samples of the first group and in six samples of the second group parvovirus-like particles were observed. Both empty and full particles were found and the diameter measured 23 k 2 nm. (n. = 29). In some fecal extracts the particles appeared aggregated, but in general they were randomly distributed over the grid.

The same stool suspensions used in the EM studies were inoculated onto MDCK cell cultures after chloroform treatment. A cytopathic agent was isolated from the stools of one dog in the breedin colony, although cytopathic in subsequent passages. Intranuclear inclusion bodies very similar to those described for the later stages of FPV infected cell cultures (9) were observed in the four subsequent passages of infected MDCK cells which had been stained with H E (Fig. 1) . Electron microscopic examination of water-disruptedy infected MDCK cells revealed the same CPV particles that were found in fecal samples. The virus, which apparently passed a 220 nm. filter, could be reisolated from the same sample.

hanges were minor and disappeared during pro P onged incubation periods and Hemagglutination by CPV and FPV antigen CPV and FPV antigens were prepared from infected MDCK (second assage) and CrFK cell cultures respectively as described above and tested For hemagglutinating activity at 4 O C with erythrocyte suspensions from different animal s ecies. The results of these tests are summarized in Table 1: antigen only caused agglutination of swine erythrocytes. Specificity of these reactions could be assessed in HA1 tests with specific antisera produced in the experimentally infected dogs and cats as described below. Erythrocytes from the other animal species were not agglutinated by CPV or FPV antigen, although some inconsistent results were obtained with rat erythrocytes. Control anti en preparations, prepared similarly from non-infected MDCK or CrFK cu P tures did not show hemagglutinating activity.

CPV antigen agg f utinated AGM, cat and swine erythrocytes, whereas FPV 

In HA1 tests the pre-inoculation sera of the experimentally infected dogs and cats, the ost-infection sera of these dogs, the post-vaccination serum of the cat (before cfallenge), the second serum of the non-vaccinated cat and the post-challenge sera of both cats were tested with the CPV and FPV antigen (Tab. 

IEM test were performed with the post-vaccination (anti-FPV) serum of the cat (before experimental infection) on fecal samples of the dog from which CPV was isolated and of another dog showing CPV particles in its stools.

With both fecal samples clear immune ag regates could be produced using binations used in the homologous and heterologous HA1 tests were tested in the IEM tests, with the exceptions shown in Table 2 . The results were essentially the same as in the HA1 tests: a two-way cross-reaction between both viruses was demonstrated in these IEM tests with the sera which were also this specific anti-FPV serum (Fig. 2) . In a dp dition the same serum-virus compositive in the HA1 test, while all presera failed to produce any aggregation (Tab. 2, Fig. 3 ).

Indirect IF tests were performed in Leighton tube MDCK and CrFK cell cultures, three days after infection with the third passage of CPV and FPV respectively, using the same serum-viral antigen combinations as used in the previous tests with the exceptions shown in Table 2 . In homologous and heterologous systems a clear, predominantly nuclear, immunofluorescence was observed (Fig. 4) in all combinations, which were also positive in ,the two previous tests. Inclusion bodies could be shown to consist of accumulations of virus-specific antigen with these tests (Fig. 4) . The negative combinations in the previous tests also proved to be negative in the corresponding IF tests (Tab. 2).

Minimal neutralization indices of sera used in the previous three tests were determined for CPV and FPV in Leighton tube cultures, using the presence of intranuclear inclusion bodies in H E stained cultures as a criterion 

In the present paper we describe the isolation in the Netherlands of a virus from dogs with acute symptoms of diarrhea and with pathological lesions very similar to those of feline panleukopenia. A close antigenic relationship between this virus and FPV, a parvovirus of the cat, could be demonstrated. On the basis of this relationship, size and morphology of the particles, resistance to chloroform and the nature of the cytopathic changes caused in cell cultures, CPV appears to be a parvovirus. Using pre-and post-inoculation sera from an FPV-vaccinated and a non-vaccinated cat and also from nonvaccinated dogs, which were all experimentally infected with CPV, we were able to demonstrate a two-way cross-reactivity between CPV and FPV in HAI, IEM, IF and S N tests. Although CPV and FPV are closely related, they agglutinate a different spectrum of erythrocytes: CPV agglutinates AGM, cat and swine erythrocytes at 4 OC and FPV only agglutinates swine probably also distinguish CPV from the previously isolated canine parvovirus MVC, which failed to show cross-reactivity with FPV in HA1 tests (15) . Another difference between CPV and FPV may be the cell spectrum in which both viruses multiply: CPV did multiply in both MDCK and CrFK cells, although higher titers Qf infectivity were found in the homologous system, whereas FPV could only be propagated in the latter (OSTERHAUS, unpublished observations). However, this could be a matter of adaptation. A practical implication of the close antigenic relationship between CPV and FPV might be the protection of dogs from CPV infections by vaccination with FPV vaccines. Apart from a mild leukopenia and teniperature rise disease symptoms resembling feline panleukopenia were absent in the non-vaccinated cat after experimental infection with cell culture propagated CPV. It is not unlikely that environmental conditions and secondary bacterial infections may be im ortant in the pathogenesis as was shown for feline panleukopenia (13). d i s is also suggested by the fact that the two dogs did not develop any overt clinical symptoms after experimental infection with CPV, although also in these animals virus multiplication was evidenced by seroconversion. However, the potential role played by this virus in the etiology of contagious diarrhea is suggested by various reports from all over the world, associating a similar virus with outbreaks of diarrhea in the dog. Moreover, in a large number of fecal samples from dogs in the Netherlands, screened during the last few years no parvovirus-like particles were observed (unpublished observations), whereas almost all stools collected from dogs involved in the outbreaks described above were positive. As in mink virus enteritis (for review see 15), a virus closely related to FPV seems to cause a canine viral enteritis. It is still an open question why infections with CPV occurred apparently suddenly in many parts of the worl'd. Serological and clinical data from the Netherlands, and also from the USA (4), suggest that infections date only from the last few years.

Parvovirus-like particles were demonstrated by negative contrast electron microscopy in feces of dogs involved in outbreaks of a contagious diarrhea in the Netherlands. The virus was isolated in a continuous cell line in which it produced intranuclear inclusion bodies. It showed a close antigenic relationship with feline panleukopenia virus: a two-way cross-reaction was observed in hemagglutination inhibition, immune electron microscopy, immunofluorescence and serum neutralization tests.

in Faces von Hunden in den Niederlanden nachgewiesen. Das Virus wurde in einer permanenten Zell-Linie isoliert, in der intranukleare Einschlufikorperchen nachgewiesen werden konnten. Es zeigte eine nahe Antigenverwandtshaft mit dem Virus der felinen Panleukopenie. Eine beiderseitige Kreuzreaktion wurde beobachtet in der Hamag lutinationshemmung, Immunelektronenmikroskopie, Immunofluoreszenz un if im Serumneutralisations-Tests.

Isolement d'un virus trks proche au virus de la panleucophie fCline chez des chiens diarhCiques Des particules ressemblant aux parvovirions ont ktk dkmontrkes au microscope klectronique (contraste-nkgatif) dans les matihres fkcales des chiens diarrhkiques aux Pays-Bas. Le virus Ctait isolk sur une ligne cellulaire continue dans laquelle il produisait des corpuscules d'inclusion intranuclkairs. Le virus manifestait un parent6 antigknique avec le virus de la panleucopknie fCline: des rkactions croiskes dans les deux sens ktaient observtks, utilisant des mkthodes d'inhibition d'hkmagglutination, immunomicroscopie klectronique, immunofluorescence et sCroneutralisation.

Resumen Aislamiento de un virus estrechamente relacionado con el virus de la panleucopenia felina en perros con diarrea Particulas semejantes a parvoviriones fueron demostrados por medio de microscopia electrbnica (contraste negativo) en muestras fecales de perros durante endemias de diarrea infecciosa en 10s Paises Bajos. Se mostrb el aislamiento del virus en una linea celular continua en donde producia corpfisculos de inclusibn intranucleares. El virus tiene una relacibn antigknica estrecha con el virus de la panleucopenia felina: reacciones cruzadas en ambos sentidos fueron observadas por m d i o de pruebas de inhibicibn de la hemaglutinacibn, inmunomicroscopia electrbnica, inmunofluorescencia y sueroneutralizacibn.

Status Report: Canine viral enteritis

Viruserkrankungen des Hundes. World Small Anim. Vet. Ass. 21th Annual Meeting

Recovery and characterization of a minute virus of canines

Parvoviral enteritis and panleukopenia in dogs

Diarrhea in puppies: Parvovirus-like particles demonstrated in their feces

Canine enteritis caused by a parvovirus-Illinois

Parvolike virus associated enteritis in dogs, morphologically resembling the enteric lesions of feline panleukopenia

A possible parvovirus associated with an epidemic gastroenteritis of dogs in Canada

Feline panleukopenia virus. 11. Some features of the cytopathic effects in feline kidney monolayers

An enteric disease of dogs resembling feline panleukopenia

Antiviral antibodies in dogs in the Netherlands

Closely Related to Feline Panleukopenia Virus from Dogs with Diarrhea 21

Seroepidemiology of feline infectious peritonitis virus infections using transmissible gastroenteritis virus as antigen

Experimental feline infectious enteritis in the germfree cat

A hemagglutination-inhibition technique for typing adenoviruses

The parvoviruses

Author's address: National Institute of Public Health

The authors wish to thank Miss M. A. MERCELINA, G. A. DROST and A. R. BEYLEVELDT for skillful technical assistance; Miss A. VAN