Novel Variant of Beilong Paramyxovirus in Rats, China

To the Editor: In 2003, two cDNA strands were identified in a human mesangial cell line during experimental screening for genes upregulated by angiotensin II (1). Sequence analysis showed that the strands were homologous to the matrix, fusion, and phosphoprotein genes of paramyxoviruses, suggesting the possibility of a novel paramyxovirus (2,3). Subsequent research found that these sequences, believed to originate from human kidney mesangial cell lines, were not amplifiable from such cell lines or human kidney samples but were amplifiable from a rat kidney mesangial cell line (4). Isolation and complete genome sequencing of the virus confirmed that it was a novel paramyxovirus of the subfamily Paramyxovirinae, named Beilong virus (BeV).

BeV is most closely related to J virus, discovered in autoculture of kidney tissue from a moribund house mouse, and Tailam virus from Sikkim rats (5,6). Because J virus and Tailam virus were found to originate in rodents and BeV was amplifiable from a rat kidney mesangial cell line, we hypothesized that BeV was a novel paramyxovirus originating in rats. To test this hypothesis, we conducted a territorywide molecular epidemiologic study of rats and other mammals to evaluate this novel paramyxovirus.

We tested 4,130 samples from 1,398 animals collected from various locations in Hong Kong, People’s Republic of China, during September 2008–August 2009 (Table). These included 480 kidney, spleen, respiratory swab, and anal swab samples from 120 asymptomatic rats (105 brown rats [Rattus norvegicus] and 15 black rats [R. rattus]). To prevent cross contamination, we used disposable scalpels, decontaminated the work surface, and used sterile gloves for each tissue sample. We performed RNA extraction and reverse transcription PCR by using strategies we previously published for discovery and epidemiologic study of paramyxoviruses (6–9).

Table. Mammals screened for Beilong virus, People’s Republic of China, September 2008–August 2009.

Animal	Sample type*
Bats, n = 502	Throat swab, rectal swab
Cats, n = 130	Nasal swab, rectal swab, urine, blood
Cattle, n = 100	Nasal swab, rectal swab, liver, buffy coat, plasma
Dogs, n = 149	Nasal swab, rectal swab, urine, blood
Hamsters, n = 49	Throat swab, intestinal swab, kidney
Pigs, n = 100	Nasal swab, rectal swab, liver, blood
Wild urban rodents, n = 120	Rectal swab, throat swab, rectal swab, kidney, spleen
Wild rural rodents, n = 248	Throat swab, rectal swab

*All sample types listed for each animal were collected except wild rodents: 237 throat swab and 246 rectal swab samples were collected from 248 wild rodents.

We performed BeV screening by PCR amplification of a 440-bp fragment of the large (L) gene, located at the 5′ end of the genome and used specific primers (LPW9739 5′-GGAGGATTCCCTCATAGAGAA-3′ and LPW9741 5′-CTCATATGTATTTACATTTAAACCA-3′). The PCR mixture (25 μL) contained cDNA, PCR buffer (10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl, 3 mmol/L MgCl₂, and 0.01% gelatin), 200 μM of each dNTP, and 1.0 units of Taq polymerase (Applied Biosystems, Foster City, CA, USA). The mixtures were amplified in 60 cycles at 94°C for 1 min, 48°C for 1 min, and 72°C for 1 min; and a final extension at 72°C for 10 min in an automated thermal cycler (Applied Biosystems).

BeV in the positive samples was confirmed by amplifying a 318-bp fragment of the nucleocapsid (N) gene of BeV, located at the 5′ end of the genome; by using specific primers (LPW10723 5′-TATATGGTTGAGATYCTNATHGA-3′ and LPW10408 5′-CCATKGCRTAGCTCCADAG-3′) and experimental conditions described above. We confirmed the specificities of the primers by testing samples positive for Tailam virus (6), which all showed negative results.

Results of reverse transcription PCR for a 440-bp fragment in the large gene of BeV were positive for 40 kidney and 9 spleen samples from 43 rats (40 brown rats and 3 black rats). Sequencing and phylogenetic analysis showed 6–13 base differences between the sequences and the corresponding region in the large gene of BeV (GenBank accession no. NC_007803), suggesting that this is a novel variant of BeV in our locality.

Results of reverse transcription PCR for a 318-bp fragment in the N gene of BeV were positive in the same 40 kidney and 9 spleen samples from the 43 rats. Sequencing and phylogenetic analysis showed 1–9 base differences between the sequences and the corresponding region in the N gene of BeV. The kidney and spleen samples were positive in 4 brown rats and 2 black rats. The L and N gene sequences amplified from the kidney and spleen samples were identical in 5 of the 6 rats. However, in 1 brown rat, L and N gene sequences from the kidney and spleen samples differed by 4 and 6 bases, respectively, suggesting the possibility of 2 strains of BeV in the same rat. None of the samples from the other mammals were positive. The authenticity of the results was supported by identical results from 2 independent genes of the BeV genome, sequence variations in the L and N genes from the positive samples, and negative results from all other mammals tested.

This study suggests that BeV and its variants are endemic in brown rats and black rats, but it is not known whether transmission is vertical or horizontal. Detection of BeV and Tailam virus in kidney and spleen samples, but not respiratory or anal swabs, suggested that they are probably systemic viruses excreted in urine. Phylogenetic and genomic evidence support the grouping of BeV, Tailam virus, and J virus into a new genus of Paramyxovirinae. Distinctly, the genomes of all 3 viruses contain 8 genes (3′-N-P/V/C-M-F-SH-TM-G-L-5′). We speculate that the ancestor of these closely related paramyxoviruses infected the common ancestor of rats and mice, with subsequent co-evolution and divergence with the host.