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
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ABSTRACT |
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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.
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INTRODUCTION |
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The group of human parainfluenzaviruses (PIVs) includes four types (14). 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.
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METHODS |
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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 ml1 (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 µl1. 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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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. ml1. 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 kg1 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).
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RESULTS |
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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Castleman, W. L., Brundage-Anguish, L. J., Kreitzer, L. & Neuenschwander, S. B. (1987). Pathogenesis of bronchiolitis and pneumonia induced in neonatal and weanling rats by parainfluenza (Sendai) virus. Am J Pathol 129, 277286.[Abstract]
Craighead, J. E., Cook, M. K. & Chanock, R. M. (1960). Infection of hamsters with para influenza 3 virus. Proc Soc Exp Biol Med 104, 301304.
Felsenstein, J. (1992). Estimating effective population size from samples of sequences: inefficiency of pairwise and segregating sites as compared to phylogenetic estimates. Genet Res 59, 139147.[Medline]
Henricks, P. A., Van Esch, B., Engels, F. & Nijkamp, F. P. (1993). Effects of parainfluenza type 3 virus on guinea pig pulmonary alveolar macrophage functions in vitro. Inflammation 17, 663675.[CrossRef][Medline]
Holmes, M. J. & Ramsay, A. J. (1988). Rabbit model for mucosal immunity in the bowel. I. Establishment of virus-infected ileal loops. J Med Virol 25, 271280.[Medline]
Ito, Y., Tsurudome, M., Hishiyama, M. & Yamada, A. (1987). Immunological interrelationships among human and non-human paramyxoviruses revealed by immunoprecipitation. J Gen Virol 68, 12891297.[Abstract]
Murphy, T. F., Dubovi, E. J. & Clyde, W. A., Jr (1981). The cotton rat as an experimental model of human parainfluenza virus type 3 disease. Exp Lung Res 2, 97109.[Medline]
Ohsawa, K., Yamada, A., Takeuchi, K., Watanabe, Y., Miyata, H. & Sato, H. (1998). Genetic characterization of parainfluenza virus 3 derived from guinea pigs. J Vet Med Sci 60, 919922.[CrossRef][Medline]
Ottolini, M. G., Porter, D. D., Blanco, J. C. G. & Prince, G. A. (2002). A cotton rat model of human parainfluenza 3 laryngotracheitis: virus growth, pathology, and therapy. J Infect Dis 186, 17131717.[CrossRef][Medline]
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, 103111.[Medline]
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406425.[Abstract]
Schmidt, N. J. & Emmons, R. W. (editors) (1989). Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections, 6th edn. Washington, DC: American Public Health Association.
Simmons, J. H., Purdy, G. A., Franklin, C. L., Trottier, P., Churchill, A. E., Russell, R. J., Besch-Williford, C. L. & Riley, L. K. (2002). Characterization of a novel parainfluenza virus, caviid parainfluenza virus 3, from laboratory guinea pigs (Cavia porcellus). Comp Med 52, 548554.[Medline]
Welch, B. G., Snow, E. J., Jr, Hegner, J. R., Adams, S. R., Jr & Quist, K. D. (1977). Development of a guinea pig colony free of complement-fixing antibodies to parainfluenza virus. Lab Anim Sci 27, 976979.[Medline]
Wyde, P. R., Amborose, M. W., Meyer, H. L. & Gilbert, B. E. (1990). Toxicity and antiviral activity of LY253963 against respiratory syncytial and parainfluenza type 3 viruses in tissue culture and in cotton rats. Antiviral Res 14, 237247.[CrossRef][Medline]
Received 6 October 2004;
accepted 1 December 2004.
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