Contamination of a specific-pathogen-free rat breeding colony with Human parainfluenzavirus type 3

Hironori Miyata1, Tamotsu Kanazawa2, Kazumoto Shibuya3 and Shigeo Hino4

1 Animal Research Center, University of Occupational and Environmental Health, 1-1 Iseigaoka Yahatanishi, Kitakyushu 807-8555, Japan
2 Department of Parasitology and Tropical Public Health, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka Yahatanishi, Kitakyushu 807-8555, Japan
3 Nippon Institute for Biological Science, Shin-machi, Ome, Tokyo 198-0024, Japan
4 Division of Virology, Faculty of Medicine, Tottori University, Yonago 683-8503, Japan

Correspondence
Hironori Miyata
h-miyata{at}med.uoeh-u.ac.jp


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Routine antibody surveillance for Sendai virus in a breeding colony suggested viral invasion into laboratory rats. A more specific haemagglutination-inhibition test implied that the agent was related closely to Human parainfluenza virus type 3 (hPIV3), rather than Sendai virus. To isolate this virus, Vero cells were inoculated with lung homogenates of 30 young animals from the colony. One of the cultures became positive at the second passage by RT-PCR directed to the hPIV3 NP and L genes. Cytopathic effect with cell fusion was observed at the third passage. The HN gene of this virus (KK24) had >93 % similarity to those of other hPIV3 isolates, suggesting a human origin of KK24. Experimental intranasal inoculation of KK24 into SD rats showed virus replication in the lungs at 3–5 days post-infection (p.i.). Pathological examination of the lungs at day 5 p.i. indicated a moderate detachment, degradation and apoptosis of bronchial epitheliocytes with peribronchial mononuclear infiltrations. At day 7 p.i., these changes became less prominent, and no lesions were apparent at day 10 p.i. or later. The infected rats seroconverted at day 7 p.i. On the contrary, none of the 30 experimentally infected ICR mice showed any pathological lesions in their lungs, despite seroconversion at 7 days p.i. These results suggest that hPIV3 can invade rat colonies and has a moderate and transient pathogenicity in rats. This is the first report of non-experimental hPIV3 infection in laboratory rats, unexpectedly detected by antibody screening for Sendai virus.

The GenBank/EMBL/DDBJ accession numbers for the hPIV3 sequences reported in this paper are AB195612 for B12 NP, AB189962 for KK24 NP, AB195610 for KK24 L, AB189960 for KK24 HN, AB195613 for BRD NP, AB195611 for BRD L and AB189961 for BRD HN.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In 2002, during a routine antibody-surveillance process, specific-pathogen-free (SPF) rats in a breeding colony were found to be positive for anti-Sendai virus antibodies by an ELISA kit. Because invasion of Sendai virus into a commercial breeding colony causes serious consequences not only for the breeder, but also for research communities (Castleman et al., 1987), the cause of this incident was scrutinized virologically. As the cross-reactive antigens among parainfluenzaviruses are well-known (Bellini et al., 1998; Ito et al., 1987), the positive sera were tested further in a more specific assay, i.e. a haemagglutination-inhibition (HI) test for detection of Human parainfluenza virus type 3 (hPIV3) using a commercially available antibody (Schmidt & Emmons, 1989). The results suggested that the virus involved is related more closely to hPIV3 than to Sendai virus.

The group of human parainfluenzaviruses (PIVs) includes four types (1–4). They belong to the order Mononegavirales, family Paramyxoviridae, subfamily Paramyxovirinae. The current taxonomy of viruses classifies these human PIVs into two separate genera, Respirovirus and Rubulovirus (http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/index.htm). While the latter includes the type 2 and 4 PIVs, the former possesses type 1 (human hPIV1 and mouse Sendai virus) and type 3 (hPIV3 and bovine PIV3, a causative agent for shipping fever) PIVs. Animals known to be infected with PIV3s other than the human and bovine species include hamster (Craighead et al., 1960), cotton rat (Murphy et al., 1981; Porter et al., 1991), guinea pig (Henricks et al., 1993) and rabbit (Holmes & Ramsay, 1988), but only under laboratory conditions. The nucleic acid similarity of the open reading frame (ORF) of the HN gene among hPIV3 sequences deposited in GenBank/EMBL/DDBJ is >93 %.

Asymptomatic outbreaks of PIV3 among laboratory guinea-pig colonies have been documented (Ohsawa et al., 1998; Simmons et al., 2002; Welch et al., 1977). These invasions were also detected by assays to detect antibodies against Sendai virus. Contamination with hPIV3 was suggested in these instances by the HI assay to detect hPIV3. Genomic analysis of the isolated virus revealed that the virus concerned clustered within hPIV3, rather than bovine PIV3 (Ohsawa et al., 1998).

These facts suggested that the virus that invaded this SPF rat colony might also be hPIV3. The rat has not been recognized as a host species for either human or rat PIV3, either in the laboratory or in the wild. In order to shed light on the incident, the virus was isolated and its taxonomic position and pathogenicity in rats and mice were defined.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells and viruses.
Vero cells were cultured in a 5 % CO2 incubator with Dulbecco's modified Eagle's medium (DMEM) (Gibco-BRL) supplemented with 10 % fetal calf serum (FCS; Filtron), 100 U penicillin ml–1 and 100 µg streptomycin ml–1. An hPIV3 strain, 65-899, was obtained as a positive control, courtesy of Dr Hiroshi Sato, Nagasaki University, Japan.

Antibody detection.
An ELISA system, Monilizer (Wakamoto Pharmaceutical), was used for anti-Sendai virus antibody detection in the surveillance of a breeding colony. The assay system has been known to cross-detect other PIVs, due to close similarities of these viruses (Bellini et al., 1998). To detect the type-specific antibody against hPIV3, an HI test kit for hPIV3 (Denka Seiken) was used.

Direct immunofluorescence, haemoadsorption and plaque assays.
A direct immunofluorescence assay (IFA) with a fluorescent anti-hPIV3 rabbit antibody (Denka Seiken) was performed to detect the hPIV3 antigens in infected Vero cells. The infected cells were harvested by trypsinization, washed several times with Dulbecco's PBS [PBS(–)], plated on a 10-well slide glass and cultured with complete medium for 16 h. After drying, the antigen plates were fixed with chilled acetone and frozen until use.

For haemoadsorption and plaque assays, Vero cell sheets were overlaid with 0·7 % LO3 agarose (TaKaRa) in DMEM and trypsin, followed by incubation for 6 days. The haemoadsorption assay was performed with 0·5 % guinea-pig erythrocytes after removing the soft agarose layer. After 30 min adsorption, cells were washed gently with PBS(–) three times. For the plaque assay, the culture was stained with 0·01 % neutral red.

PIV3 isolation.
We isolated the virus directly from rats within the breeding colony. Monolayered Vero cells in a 35 mm dish were inoculated with 200 µl of a 10 % lung homogenate. After adsorption at 34 °C for 1 h, the cells were fed with 3 ml DMEM without FCS, but containing 25 U trypsin ml–1 (Sigma), and incubated for 5 days at 34 °C. The supernatant was passed blindly onto fresh Vero monolayers. On day 4, the supernatant was passed to the tertiary culture. Aliquotted supernatants on day 5 were freeze-stocked at –80 °C until use.

Extraction of nucleic acids and amplification by RT-PCR.
RNA was extracted with an RNeasy kit (Qiagen) from 100 µl of each 10 % lung homogenate or tissue-culture supernatant. The RNA was dissolved in nuclease-free distilled water at a concentration of 0·1 µg µl–1. Primers for PIV3 detection (Table 1) were designed based on a published hPIV3 sequence (GenBank accession no. AB012132). Although the virus had been recovered from a guinea pig, its sequence identity to other reported hPIV3s revealed that the virus was of human origin. While the sequence identity of the HN ORF to those of other hPIV3s is >93 %, its identity to those of bovine PIV3, human and mouse PIV1s and SV5 (a simian PIV2) is 75, 57, 55 and 47 %, respectively.


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Table 1. Primer sequences and positions used in PIV3 RT and long RT-PCR

Base positions are according to an hPIV3 sequence (GenBank accession no. AB012132).

 
A 10 µl aliquot of RNA was subjected to short diagnostic RT-PCRs directed to the NP and L genes by using a Titan One-Tube RT-PCR kit (Roche Diagnostics). A 5 µl aliquot of the first PCR product was added to the nested PCR with the inner primer pair. After reverse transcription for 30 min at 50 °C, both the first and nested PCR steps consisted of 30 cycles of 30 s at 94 °C (2 min on the first cycle), 30 s at 52 °C and 45 s at 72 °C. The amplified products were electrophoresed on a composite gel of 2 % NuSieve GTG agarose (FMC Bioproducts) and 1 % LO3 agarose.

To amplify the whole HN gene of the virus-culture supernatant, a single-step long RT-PCR was used with the PIV6750 and PIV8563 primers (Table 1) and with Tbr EXT DNA polymerase (Finzymes). Following reverse transcription for 30 min at 50 °C, the PCR step consisted of 30 cycles of 30 s at 94 °C (2 min on the first cycle), 45 s at 50 °C (an additional 5 s on each cycle) and 2 min at 72 °C. The amplified products were electrophoresed on a 1 % LO3 agarose gel.

Preparation of a PIV3 fraction from dust in the breeding room.
To demonstrate the possibility of hovering viral aerosol in the breeding room, we planned to amplify the PIV3 HN gene from the room dust. A 20 g sample of floor dust in the breeding room was suspended in 500 ml PBS(–) for 20 min at room temperature with continuous magnetic stirring. After filtering through a sterile cotton mesh, the solution was centrifuged at 5000 r.p.m. for 10 min at 4 °C. The filtrate through a prefilter (AP15004700; Millipore) was ultracentrifuged at 100 000 g for 2 h at 4 °C using an SW28 rotor (Beckman Coulter). The final pellets were suspended in 2 ml DMEM and used as the RNA source for RT-PCR. Because of the small amount of viral RNA in the dust samples, a nested RT-PCR was employed: the PIV6721 and PIV8633 primers were used for the reverse transcription and first PCR steps, and the PIV6750 and PIV8563 primers were used for the nested step of PCR (Table 1).

Determination and sequence analysis of the PIV3 HN gene.
The products amplified by RT-PCR were cloned into the EcoRV site of pBluescript after 5'-end repair by the Klenow fragment (TaKaRa) and phosphorylation of the products' 5' ends by polynucleotide kinase (TaKaRa). Both strands were sequenced with a BigDye Terminator Cycle Sequencing ready reaction kit on an ABI PRISM 3100 genetic analyser (both from Applied Biosystems). Sequence analysis was performed by using GENETYX (Mac). Sequence alignments were generated by the DDBJ version of CLUSTAL W. Phylogenetic trees were constructed by the neighbour-joining method (Saitou & Nei, 1987). The reliability of the phylogenetic results was assessed by using 1000 bootstrap replications (Felsenstein, 1992). The final tree was obtained by using the TREEVIEW program, version 1.6.6.

Experimental infection.
The supernatant of Vero cells infected with the isolated virus, KK24, was used as the virus stock: the titre of the virus stock was 4·0x107 p.f.u. ml–1. Thirty each of outbred 6-week-old female SPF IGS SD rats and CD-1 mice, all free from the anti-PIV3 antibody, were purchased from Charles River, Japan. The SD rats were inoculated intranasally with 40 µl KK24, containing either 1·6x106 or 1·6x103 p.f.u. CD-1 mice were inoculated with 10 µl, containing either 4·0x105 or 4·0x102 p.f.u. Two animals each were sacrificed under anaesthesia with a mixture of 75 mg ketamine and 1 mg medetomidine kg–1 by intraperitoneal injection and their lungs were resected at days 0, 3, 5, 7, 10, 14 and 21 post-infection (p.i.). The trachea and right lung were fixed with 10 % formalin for pathological examinations. The left lung was used for 10 (w/v) % homogenate in DMEM and antibiotics without FCS. All animal experiments were performed under the control of the Ethics Committee of Animal Care and Experimentation in accordance with the Guiding of Principle for Animal Care Experimentation, University of Occupational and Environmental Health, Japan and the Japanese Law for Animal Welfare and Care (no. 221).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Sensitivity of RT-PCR
Two sets of diagnostic short RT-PCRs, directed to the NP and L genes of hPIV3, were used to cover the distant areas of the genome. Sensitivities of the RT-PCRs were calibrated with RNA samples extracted from the pretitrated 65-899 strain of hPIV3 grown in Vero cells. The sensitivities of the one-step RT-PCRs for the NP and L genes, thus calibrated, were 10 000 and 1000 p.f.u. RNA equivalents per reaction, respectively. Those of the nested RT-PCRs for the NP and L genes were both 2·5 p.f.u. RNA equivalents per reaction (data not shown).

Virus isolation
To isolate virus from rats within the colony, antibody titres of young animals born to seropositive dams were tested by the HI test for hPIV3 (data not shown). From the waning curve of the maternal antibody titres, rats at the age of 7 weeks were chosen as the candidate source tissues for virus isolation. For example, the antibody prevalence and mean HI titres of five rats at 6, 7, 8 and 9 weeks after delivery were 5/5 (1/12·8), 3/5 (1/4·8), 4/5 (1/6·4) and 5/5 (1/19·2), respectively. Among the lung extracts of 30 young female rats, five gave a positive signal at least in one of the two nested short RT-PCRs; only B12 had a weakly detectable amount of HI antibody against hPIV3. While samples B14, B24 and B28 were positive in both nested RT-PCRs directed to the NP and L genes, samples B12 and B19 were positive only in the RT-PCR directed to the NP gene (Fig. 1). Direct sequencing of these PCR products showed that the NP gene of B14 was 1 nt different from those of B24 and B28, but the L genes of these three isolates were identical. The NP genes of B12 and B19 were the same as that of B14.



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Fig. 1. Nested RT-PCR of RNA extracted from the lung homogenates of 30 7-week-old rats in the breeder's colony. Only five animals out of 30 gave positive signals at least in one assay of the nested RT-PCRs directed to the NP and L genes. The reciprocals of HI titre of each animal against hPIV3 are shown at the bottom. Upper panel, NP gene; lower panel, L gene. P, Positive control, equivalent to 5 p.f.u. hPIV3 RNA; N, negative control without template; B12–B28, five rats obtained from the breeding colony; M, marker.

 
Each of these five lung homogenates was inoculated into Vero cells for virus isolation. On day 5, the supernatant was passed blindly onto fresh Vero cells. On day 4 of the secondary culture, the B24 culture supernatant turned positive in the one-step RT-PCR directed to the L gene (Fig. 2a). The same sample was positive in the nested RT-PCRs directed to both the NP and L genes (Fig. 2b, d). On day 3 of the third passage, cell fusion was observed in the B24 culture (Fig. 3a). A specific direct fluorescence assay with the anti-hPIV3 antibody on the same day revealed strong signals in the cytoplasm of Vero cells (Fig. 3b). An HI-positive serum of a naturally infected rat also revealed strong reactivity against the intracytoplasmic antigens by indirect IFA (data not shown). Infectious centres could be seen by haemoadsorption using guinea-pig erythrocytes (Fig. 3c) and distinct plaques appeared on the Vero cell sheet (Fig. 3d). The culture supernatant contained 5x107 p.f.u. virus ml–1. The virus was designated strain KK24. The cultures of the other four samples were not positive in any of the nested RT-PCRs or by other virological markers.



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Fig. 2. RT-PCR of RNA extracted from the culture fluid of Vero cells inoculated with the lung homogenates of PCR-positive rats. The culture fluid was passed blindly once onto fresh Vero cells and the culture fluid on day 4 was used for RNA extraction. RT-PCRs were directed to the NP (a, b) and L (c, d) genes and the results of first-round (a, c) and nested (b, d) PCR are shown. Discriminators for samples are given in the legend to Fig. 1. Note that the amount of positive control, 5 p.f.u. equivalent hPIV3 RNA, is adequate for the nested PCR (b, d), but far below the detection level of the single-step PCR (a, c).

 


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Fig. 3. Isolation of virus from the lung homogenate of rat B24 in Vero cells in the presence of trypsin. (a) Cell fusion observed at day 4 of tertiary culture. (b) Direct immunofluorescence with the anti-hPIV3 rabbit serum specified in the kit. (c) Haemoadsorption infectious-centre assay with guinea-pig erythrocytes. (d) Plaque formation on Vero cells.

 
Cloning of the PIV3 HN region from the isolated virus and dust by long RT-PCR.
The supernatant at the third passage of the B24 culture gave a positive signal in the one-step RT-PCR targeted toward the HN gene, at the expected size of 1·9 kb (data not shown). The breeding-room dust extract (BRD) also showed a positive signal in the nested HN RT-PCR at the expected size of 1·9 kb (data not shown). This direct detection of the HN sequence from the BRD is considered real, because the PCRs directed to the NP and L genes were also positive and none of the sequences of the NP, L and HN genes were identical to those obtained from rat samples. Furthermore, BRD samples obtained similarly from rooms without infected rats were exclusively negative in the PCRs. These DNA products were cloned into the vector pBluescript. Both of them, KK24 and BRD, had an ORF with 1716 bp, encoding 572 aa, without any loss or addition of bases in comparison to most reported HN sequences. The nucleotide similarity of the HN ORF between KK24 and BRD was 97·6 % and that with other hPIV3s was >93 %, in comparison to 75, 57 and 55 % against bPIV3, hPIV1 and Sendai virus, respectively. Phylogenetic analysis revealed that the HN sequence of KK24 and BRD was located within the branches of hPIV3 and was related distantly to other respiroviruses, including bPIV3, hPIV1 and Sendai virus, and to rubuloviruses, including SV5 (Fig. 4a, b). A similar branching pattern was obtained with the partial NP gene sequences of hPIV3, bPIV3 and Sendai virus (Fig. 4c). These results suggest that strain KK24 is an hPIV3, rather than a rat PIV3 per se.



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Fig. 4. (a) Split view of HN ORF sequences among KK24 and BRD in relation to other hPIV3s. (b) Split view of HN ORF sequences among hPIV3s and other viruses, including bovine PIV3s, Sendai virus and SV5. (c) Split view of NP ORF sequences among representative PIVs. Asterisks in (a) and (b) show the positions of nodes that divide hPIV3s from bovine PIV3s. The scale in (a) is 10 times larger than that in (b) and (c).

 
Experimental infection.
To clarify the pathogenesis of KK24, SD rats were infected intranasally at two different doses (1·6x106 or 1·6x103 p.f.u.). Viral growth in the lung homogenates and antibody induction were monitored by RT-PCR directed to the L gene, plaque assay, IFA and HI test. In SD rats, the nested RT-PCR became positive from 3 days p.i., both in the high- and low-dose groups (Table 2). The virus could be isolated by using a classic virological culture method at 3 and 5 days p.i., at a titre of 7·5x103–1·0x105 (g wet lung tissue)–1. These results suggested that KK24 could replicate in rat lungs after intranasal inoculation. Anti-hPIV3 antibody first became detectable at 7 days p.i. by indirect IFA and HI, and increased its intensity until day 21 p.i. No infectious virus could be recovered from any animals with the antibody detected by either IFA or HI. However, the highly sensitive nested RT-PCR detected the signal until day 21 p.i.


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Table 2. Intranasal infection of KK24 into rats

 
By gross observations, none of these rats with detectable antibodies showed abnormalities in their lungs. Histological examinations on pulmonary sections obtained on days 0 and 3 were normal. However, both rats sampled on day 5 p.i. showed mild to moderate pathological lesions in bronchus and peribronchiolar areas, including degeneration, apoptosis and desquamation of bronchial epithelium (Fig. 5). The lamina propria of bronchial mucosa and the peribronchial regions were infiltrated by mononuclear cells associated with oedema and degenerations. Intranuclear eosinophilic inclusion bodies were also observed. On day 7 p.i., one of two animals was free from apparent histological changes and another had milder lesions than those seen on day 5 p.i. None of the animals on days 10 and 12 showed significant histopathological abnormalities. The pathological changes in animals with the low challenge dose were basically the same as those with the high challenge dose; however, the intensity was less severe.



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Fig. 5. Haematoxylin and eosin staining of the lung of rat R10 at 5 days p.i., inoculated with 1·6x106 p.f.u. KK24. (a) Peribronchial infiltration by mononuclear cells, associated with ablation of bronchial epithelium into the bronchial space (x100). (b) High magnification of (a) (x400).

 
Because of size differences of the animals, intranasal infection of KK24 into CD-1 mice resulted in a one-quarter dose in comparison to the rats, i.e., 4·0x105 or 4·0x102 p.f.u. of KK24 intranasally. Antibodies were detectable starting from 7 days p.i., but the virus never became detectable by direct titration on Vero cells (Table 3). The highly sensitive nested RT-PCR detected the positive signal in one animal each at days 3 and 5 p.i., both in the low-challenge-dose group. None of the mice in this experiment showed any gross or histological abnormalities in the pathological examinations.


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Table 3. Intranasal infection of KK24 into mice

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
An antibody against Sendai virus was found in one of the periodical check-ups of rats in a breeding colony. However, a more specific HI test for hPIV-3 suggested that the contaminating virus was hPIV-3, rather than Sendai virus. A virus, KK24, was isolated from a lung homogenate obtained from one of 30 7-week-old animals in the colony. The virus showed cytopathic effect in Vero cells, including cell fusions at the tertiary passage. However, the source of KK24 was still uncertain at this stage, i.e. whether it was of human, bovine or rat origin.

Nevertheless, because the antibody in these rats reacted in an HI test that was specific for hPIV3, a reasonable similarity between KK24 and hPIV3 was expected. To set up diagnostic RT-PCR systems, a complete genomic sequence of hPIV3 (GenBank accession no. AB012132) was selected as the prototype of hPIV3, partially because it was the only full-size Japanese hPIV3 in GenBank/EMBL/DDBJ, considering the well-known geographical diversity of PIVs (Bellini et al., 1998). The virus was originally isolated from a guinea pig, but sequence studies revealed that the virus was really a human virus, rather than a guinea-pig virus (Ohsawa et al., 1998). The culture supernatant of the second passage of Vero cells inoculated with the lung homogenate of the B24 rat gave a positive signal in the short nested RT-PCRs that were directed to the hPIV3 NP and L genes.

The HN gene was selected for cloning because the data available in GenBank/EMBL/DDBJ were more abundant for this gene. The HN ORF of KK24 was 1716 bp, coding for 572 aa, as in the case of most hPIV3s. The exception was the sequence with accession no. AB012132, where the termination codon (TAA in other hPIV3s) was point-mutated to CAA, and it uses another TAA appearing 6 bp downstream. The sequence TAATCATAATTAACC was conserved in all hPIV3 sequences retrieved in this study: only AB012132 had a T to C conversion at the first T. Sequence similarities of the KK24 HN ORF to other reported PIVs revealed that KK24 is really an isolate of hPIV3, rather than a virus infecting rat species for an extended period. This is because the similarities to other hPIV3s are at least 93 %, in contrast to 75 % similarity to bovine PIV3 and 57 and 55 % similarity to human and murine PIV1, respectively.

This is, as far as we know, the first isolation of PIV3 from laboratory rats. However, KK24 was found to be a human virus, as in the case of GenBank accession no. AB012132, which was isolated from a laboratory guinea-pig colony. This suggests that the rats could have been contaminated with hPIV3 by animal caretakers in the breeding room. Because KK24 is not widely diverged from other hPIV3s, these viruses are probably relatively new to this animal colony. To avoid these unwanted contaminations, a more stringent effort for infection control is necessary in the breeding room. Vaccination strategies to cover animal caretakers have not been available for hPIV3.

The direct detection of hPIV3 from room dust by RT-PCR may suggest a new direction for the routine surveillance of contamination in breeding facilities. The potential strength of this method includes the high sensitivity of the assay, ability to survey a wide spectrum of viruses, rapid diagnosis and relative easiness for outsourcing. Although the cost of surveillance is not negligible, the potential cost of the confirmation of eradication should be much higher for the breeder than that of the screening assays in the case of contamination.

The pathogenicity of hPIV3 for laboratory rats has never been reported. The experimental infection in this report revealed the mild and transient, but distinct, pathogenicity of KK24 in rats. Pathological examinations after experimental infection revealed that lesions in the bronchus appeared on day 5 p.i., but became less prominent on day 7 and disappeared spontaneously by day 10. Unfortunately, we could not obtain an appropriate anti-PIV3 antibody to substantiate these findings immunohistochemically. Thirty uninoculated rats within the breeding room were examined histologically, but no lesions were found (data not shown), probably because of the short lives of pathological changes after the initial infection. Because young rats were infected spontaneously in this colony, the virus could have been sustained in the breeding room, at least for a while.

Two factors (continuous supply of newborns and their staying in the breeding room until disappearance of the maternal antibody) are probably key to the survival of PIV3 in the breeding colony. To support this concept, almost all of the retiring animals were found to be antibody-positive. If this concept was true, stopping mating for a few months would eradicate PIV3 from a breeding room. However, the presence of persistent spreaders could not be excluded. Cotton rats have been a good animal model for human PIV3 infection (Ottolini et al., 2002). As the viral titres in the lungs of SD rats were comparable to those reported in cotton rats (Wyde et al., 1990), SD rats might also be another appropriate animal model for hPIV3 infection.

With respect to serodiagnosis of animals in breeding colonies, some ELISA kits may pick up viruses other than the target virus with their cross-reactivity. Because it will take some time before the true nature of the agent in question is elucidated, the screening results should be handled carefully, in order not to cause confusion for both breeders and users.


   ACKNOWLEDGEMENTS
 
We thank T. Akiyoshi for technical assistance. This work was supported in part by grants-in-aid (14580803) from the Ministry of Education, Science, Sports and Culture of Japan.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bellini, W. J., Rota, P. A. & Anderson, L. J. (1998). Paramyxoviruses. In Topley & Wilson's Microbiology and Microbial Infections, 9th edition, pp. 435–461. Edited by B. W. J. Mahy & L. Collier. London: Arnold.

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Porter, D. D., Prince, G. A., Hemming, V. G. & Porter, H. G. (1991). Pathogenesis of human parainfluenza virus 3 infection in two species of cotton rats: Sigmodon hispidus develops bronchiolitis, while Sigmodon fulviventer develops interstitial pneumonia. J Virol 65, 103–111.[Medline]

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Received 6 October 2004; accepted 1 December 2004.



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