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).

. 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 amplifi able from such cell lines or human kidney samples but were amplifi able from a rat kidney mesangial cell line (4). Isolation and complete genome sequencing of the virus confi rmed 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 amplifi able 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 All material published in Emerging Infectious Diseases is in the public domain and may be used and reprinted without special permission; proper citation, however, is required. 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).
We performed BeV screening by PCR amplifi cation of a 440bp fragment of the large (L) gene, located at the 5′ end of the genome and used specifi c primers (LPW9739 G G A G G AT T C C C T C ATA G A G AA-3′ and LPW9741 5′-CTCATAT GTATTTACATTTAAACCA-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 MgCl 2 , 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 amplifi ed in 60 cycles at 94°C for 1 min, 48°C for 1 min, and 72°C for 1 min; and a fi nal extension at 72°C for 10 min in an automated thermal cycler (Applied Biosystems).
BeV in the positive samples was confi rmed by amplifying a 318-bp fragment of the nucleocapsid (N) gene of BeV, located at the 5′ end of the genome; by using specifi c primers (LPW10723 5′-TATATGGTTGAGAT YCTNATHGA-3′ and LPW10408 5′ -CCATKGCRTAGCTCCADAG-3′) and experimental conditions described above. We confi rmed the specifi cities 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 amplifi ed 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.  To the Editor: Approximately 100,000 persons worldwide have received cochlear implants for hearing loss, and more children now receive them than ever (1). Such children have a >30-fold increased risk for pneumococcal meningitis than the background rate (1,2). During 2006-2010, children born in the United Kingdom were offered the 7-valent pneumococcal conjugate vaccine (PCV7) at 2, 4, and 13 months of age (3). Those at high risk for invasive pneumococcal disease (IPD) were additionally offered the 23-valent pneumococcal polysaccharide vaccine (PPV23) at 2-5 years (3). We describe a fully vaccinated child with a cochlear implant in whom recurrent pneumococcal meningitis developed, caused by a vaccine serotype (i.e., vaccine failure). The child continues to have nonprotective antibody concentrations against the infecting serotype, despite further pneumococcal vaccination.
A previously healthy, appropriately vaccinated 23-month-old girl (Table) had a cochlear device implanted in the right ear after receiving (through the universal newborn hearing screening program) a diagnosis of profound, bilateral, sensorineural deafness. Two weeks later, she exhibited fever, lethargy, and drowsiness. On hospital admission, she had a peripheral blood leukocyte count of 19.3 × 10 9 cells/L, a neutrophil count of 17.0 × 10 9 cells/L, and C-reactive protein level 75 mg/L. Meningitis was diagnosed, and she received intravenous ceftriaxone but was too ill for a lumbar puncture. Blood cultures subsequently grew fully sensitive Streptococcus pneumoniae, later confi rmed as serotype 4 by the national reference laboratory. She was discharged after 14 days of receiving intravenous antimicrobial drugs without complications.
At 24 months, she received a fourth dose of PCV7. Blood tests 1 month later showed good antibody responses to 6 PCV7 serotypes but not to serotype 4, which did not reach the putative protective level of >0.35 μg/ mL antibody threshold (Table). At 28 months, she received 1 dose of PPV23 per national guidelines (3). Four months later, she was brought to the hospital with fever, rigors, drowsiness, and vomiting. Blood tests showed a leukocyte count of 24.4 × 10 9 cells/L, neutrophil count of 21.6 × 10 9 cells/L, and C-reactive protein level of 272 mg/L. Lumbar puncture performed the next day showed 890 leukocytes/ mL (predominantly polymorphs), cerebrospinal fl uid glucose level <1.1 mmol/L, protein level of 1.0 g/L, gram-positive diplococci on Gram staining, and positive PCR results for pneumococci, although cerebrospinal fl uid culture was negative.
A blood culture grew fully sensitive S. pneumoniae, also confi rmed by the national reference laboratory as serotype 4. She recovered after receiving intravenous ceftriaxone and oral rifampin for 2 weeks, followed by 4 weeks of oral amoxicillin and rifampin. She then received prophylactic oral penicillin for maintenance. Subsequently, an abdominal ultrasound confi rmed the presence of a spleen, and her immunoglobulin concentrations were in the normal range. At 35 months, she received another dose of PCV7, and a blood test 1 month later showed variable but high responses to 6 of the PCV7 serotypes and no response to serotype 4 (Table). Moreover, nasopharyngeal swab specimens, obtained when the patient was 39 months old and receiving penicillin prophylaxis, were positive for serotype 4.