Rachel Y. Y. Fan

Feline morbillivirus, a previously undescribed paramyxovirus associated with tubulointerstitial nephritis in domestic cats

We describe the discovery and isolation of a paramyxovirus, feline morbillivirus (FmoPV), from domestic cat (Felis catus). FmoPV RNA was detected in 56 (12.3%) of 457 stray cats (53 urine, four rectal swabs, and one blood sample) by RT-PCR. Complete genome sequencing of three FmoPV strains showed genome sizes of 16,050 bases, the largest among morbilliviruses, because of unusually long 5′ trailer sequences of 400 nt. FmoPV possesses identical gene contents (3′-N-P/V/C-M-F-H-L-5′) and is phylogenetically clustered with other morbilliviruses. IgG against FmoPV N protein was positive in 49 sera (76.7%) of 56 RT-PCR–positive cats, but 78 (19.4%) of 401 RT-PCR–negative cats (P < 0.0001) by Western blot. FmoPV was isolated from CRFK feline kidney cells, causing cytopathic effects with cell rounding, detachment, lysis, and syncytia formation. FmoPV could also replicate in subsequent passages in primate Vero E6 cells. Infected cell lines exhibited finely granular and diffuse cytoplasmic fluorescence on immunostaining for FmoPV N protein. Electron microscopy showed enveloped virus with typical “herringbone” appearance of helical N in paramyxoviruses. Histological examination of necropsy tissues in two FmoPV-positive cats revealed interstitial inflammatory infiltrate and tubular degeneration/necrosis in kidneys, with decreased cauxin expression in degenerated tubular epithelial cells, compatible with tubulointerstitial nephritis (TIN). Immunohistochemical staining revealed FmoPV N protein-positive renal tubular cells and mononuclear cells in lymph nodes. A case-control study showed the presence of TIN in seven of 12 cats with FmoPV infection, but only two of 15 cats without FmoPV infection (P < 0.05), suggesting an association between FmoPV and TIN.

Isolation and Characterization of a Novel Betacoronavirus Subgroup A Coronavirus, Rabbit Coronavirus HKU14, from Domestic Rabbits

ABSTRACT We describe the isolation and characterization of a novel Betacoronavirus subgroup A coronavirus, rabbit coronavirus HKU14 (RbCoV HKU14), from domestic rabbits. The virus was detected in 11 (8.1%) of 136 rabbit fecal samples by reverse transcriptase PCR (RT-PCR), with a viral load of up to 108 copies/ml. RbCoV HKU14 was able to replicate in HRT-18G and RK13 cells with cytopathic effects. Northern blotting confirmed the production of subgenomic mRNAs coding for the HE, S, NS5a, E, M, and N proteins. Subgenomic mRNA analysis revealed a transcription regulatory sequence, 5′-UCUAAAC-3′. Phylogenetic analysis showed that RbCoV HKU14 formed a distinct branch among Betacoronavirus subgroup A coronaviruses, being most closely related to but separate from the species Betacoronavirus 1. A comparison of the conserved replicase domains showed that RbCoV HKU14 possessed <90% amino acid identities to most members of Betacoronavirus 1 in ADP-ribose 1″-phosphatase (ADRP) and nidoviral uridylate-specific endoribonuclease (NendoU), indicating that RbCoV HKU14 should represent a separate species. RbCoV HKU14 also possessed genomic features distinct from those of other Betacoronavirus subgroup A coronaviruses, including a unique NS2a region with a variable number of small open reading frames (ORFs). Recombination analysis revealed possible recombination events during the evolution of RbCoV HKU14 and members of Betacoronavirus 1, which may have occurred during cross-species transmission. Molecular clock analysis using RNA-dependent RNA polymerase (RdRp) genes dated the most recent common ancestor of RbCoV HKU14 to around 2002, suggesting that this virus has emerged relatively recently. Antibody against RbCoV was detected in 20 (67%) of 30 rabbit sera tested by an N-protein-based Western blot assay, whereas neutralizing antibody was detected in 1 of these 20 rabbits.

Recent Transmission of a Novel Alphacoronavirus, Bat Coronavirus HKU10, from Leschenault's Rousettes to Pomona Leaf-Nosed Bats: First Evidence of Interspecies Transmission of Coronavirus between Bats of Different Suborders

ABSTRACT Although coronaviruses are known to infect various animals by adapting to new hosts, interspecies transmission events are still poorly understood. During a surveillance study from 2005 to 2010, a novel alphacoronavirus, BatCoV HKU10, was detected in two very different bat species, Ro-BatCoV HKU10 in Leschenaults rousettes (Rousettus leschenaulti) (fruit bats in the suborder Megachiroptera) in Guangdong and Hi-BatCoV HKU10 in Pomona leaf-nosed bats (Hipposideros pomona) (insectivorous bats in the suborder Microchiroptera) in Hong Kong. Although infected bats appeared to be healthy, Pomona leaf-nosed bats carrying Hi-BatCoV HKU10 had lower body weights than uninfected bats. To investigate possible interspecies transmission between the two bat species, the complete genomes of two Ro-BatCoV HKU10 and six Hi-BatCoV HKU10 strains were sequenced. Genome and phylogenetic analyses showed that Ro-BatCoV HKU10 and Hi-BatCoV HKU10 represented a novel alphacoronavirus species, sharing highly similar genomes except in the genes encoding spike proteins, which had only 60.5% amino acid identities. Evolution of the spike protein was also rapid in Hi-BatCoV HKU10 strains from 2005 to 2006 but stabilized thereafter. Molecular-clock analysis dated the most recent common ancestor of all BatCoV HKU10 strains to 1959 (highest posterior density regions at 95% [HPDs], 1886 to 2002) and that of Hi-BatCoV HKU10 to 1986 (HPDs, 1956 to 2004). The data suggested recent interspecies transmission from Leschenaults rousettes to Pomona leaf-nosed bats in southern China. Notably, the rapid adaptive genetic change in BatCoV HKU10 spike protein by ∼40% amino acid divergence after recent interspecies transmission was even greater than the ∼20% amino acid divergence between spike proteins of severe acute respiratory syndrome-related Rhinolophus bat coronavirus (SARSr-CoV) in bats and civets. This study provided the first evidence for interspecies transmission of coronavirus between bats of different suborders.

Novel Betacoronavirus in Dromedaries of the Middle East, 2013

In 2013, a novel betacoronavirus was identified in fecal samples from dromedaries in Dubai, United Arab Emirates. Antibodies against the recombinant nucleocapsid protein of the virus, which we named dromedary camel coronavirus (DcCoV) UAE-HKU23, were detected in 52% of 59 dromedary serum samples tested. In an analysis of 3 complete DcCoV UAE-HKU23 genomes, we identified the virus as a betacoronavirus in lineage A1. The DcCoV UAE-HKU23 genome has G+C contents; a general preference for G/C in the third position of codons; a cleavage site for spike protein; and a membrane protein of similar length to that of other betacoronavirus A1 members, to which DcCoV UAE-HKU23 is phylogenetically closely related. Along with this coronavirus, viruses of at least 8 other families have been found to infect camels. Because camels have a close association with humans, continuous surveillance should be conducted to understand the potential for virus emergence in camels and for virus transmission to humans.

The complete genome and proteome of Laribacter hongkongensis reveal potential mechanisms for adaptations to different temperatures and habitats.

Laribacter hongkongensis is a newly discovered Gram-negative bacillus of the Neisseriaceae family associated with freshwater fish–borne gastroenteritis and travelers diarrhea. The complete genome sequence of L. hongkongensis HLHK9, recovered from an immunocompetent patient with severe gastroenteritis, consists of a 3,169-kb chromosome with G+C content of 62.35%. Genome analysis reveals different mechanisms potentially important for its adaptation to diverse habitats of human and freshwater fish intestines and freshwater environments. The gene contents support its phenotypic properties and suggest that amino acids and fatty acids can be used as carbon sources. The extensive variety of transporters, including multidrug efflux and heavy metal transporters as well as genes involved in chemotaxis, may enable L. hongkongensis to survive in different environmental niches. Genes encoding urease, bile salts efflux pump, adhesin, catalase, superoxide dismutase, and other putative virulence factors—such as hemolysins, RTX toxins, patatin-like proteins, phospholipase A1, and collagenases—are present. Proteomes of L. hongkongensis HLHK9 cultured at 37°C (human body temperature) and 20°C (freshwater habitat temperature) showed differential gene expression, including two homologous copies of argB, argB-20, and argB-37, which encode two isoenzymes of N-acetyl-L-glutamate kinase (NAGK)—NAGK-20 and NAGK-37—in the arginine biosynthesis pathway. NAGK-20 showed higher expression at 20°C, whereas NAGK-37 showed higher expression at 37°C. NAGK-20 also had a lower optimal temperature for enzymatic activities and was inhibited by arginine probably as negative-feedback control. Similar duplicated copies of argB are also observed in bacteria from hot springs such as Thermus thermophilus, Deinococcus geothermalis, Deinococcus radiodurans, and Roseiflexus castenholzii, suggesting that similar mechanisms for temperature adaptation may be employed by other bacteria. Genome and proteome analysis of L. hongkongensis revealed novel mechanisms for adaptations to survival at different temperatures and habitats.

Identification and characterization of bocaviruses in cats and dogs reveals a novel feline bocavirus and a novel genetic group of canine bocavirus.

We report the identification and genome characterization of a novel bocavirus, feline bocavirus (FBoV), and novel bocaviruses closely related to canine bocavirus (CBoV) strain Con-161 in stray cats and dogs in Hong Kong, respectively. FBoV was detected by PCR in 7.2, 0.3, 1.6, 2.0 and 0.8% of faecal, nasal, urine, kidney and blood samples, respectively, from 364 cats, while CBoV was detected in 4.6, 5.1, 6.3 and 0.3% of faecal, nasal, urine and blood samples, respectively, from 351 dogs. Three FBoV genomes sequenced revealed the presence of three ORFs characteristic of bocaviruses. Phylogenetic analysis showed that FBoVs were related only distantly to other bocaviruses, forming a distinct cluster within the genus, with ≤ 5.7% nucleotide identities to the genome of minute virus of canines. The four CBoV genomes sequenced shared 87.4-89.2% nucleotide identities with that of CBoV strain Con-161. In addition to the three bocavirus ORFs, they encoded an additional ORF, ORF4, immediately downstream of the ORF for non-structural protein 1 (NS1), which was not found in other bocaviruses including CBoV strain Con-161. They also possessed a putative second exon encoding the C-terminal region of NS1 and conserved RNA-splicing signals, previously described in human bocaviruses. Partial VP1/VP2 sequence analysis of 23 FBoV and 25 CBoV strains demonstrated inter-host genetic diversity, with two potential genetic groups of FBoV and a novel CBoV group, CBoV-HK, distinct from the three groups, CBoV-A to -C, found in the USA. Although the pathogenicity of FBoV and CBoV remains to be determined, their presence in different host tissues suggested wide tissue tropism.

Rediscovery and genomic characterization of bovine astroviruses

The genus Mamastrovirus belongs to the family Astroviridae and consists of at least six members infecting different mammalian hosts, including humans, cattle and pigs. In recent years, novel astroviruses have been identified in other mammalian species like roe deer, bats and sea lions. While the bovine astrovirus was one of the earliest astroviruses to have been studied, no further research has been performed recently and its genome sequence remains uncharacterized. In this report, we describe the detection and genomic characterization of astroviruses in bovine faecal specimens obtained in Hong Kong. Five of 209 specimens were found to be positive for astrovirus by RT-PCR. Two of the positive specimens were found to contain sequences from two different astrovirus strains. Complete genome sequences of approximately 6.3 kb in length were obtained for four strains, which showed similar organization of the genome compared to other astroviruses. Phylogenetic analysis confirmed their identities as members of the genus Mamastrovirus, and showed them to be most closely related to the Capreolus capreolus astrovirus. Based on the pairwise genetic distances among their full-length ORF2 sequences, these bovine astroviruses may be assigned into at least three different genotype species. Sequence analysis revealed evidence of potential recombination in ORF2. In summary, we report the first genome sequences of bovine astroviruses and clearly establish the species status of the virus. Additionally, our study is among the first to report co-infection by different astrovirus genotypes in the same host, which is an essential step for recombination to occur.

Identification of a novel feline picornavirus from the domestic cat

ABSTRACT While picornaviruses are known to infect different animals, their existence in the domestic cat was unknown. We describe the discovery of a novel feline picornavirus (FePV) from stray cats in Hong Kong. From samples from 662 cats, FePV was detected in fecal samples from 14 cats and urine samples from 2 cats by reverse transcription-PCR (RT-PCR). Analysis of five FePV genomes revealed a distinct phylogenetic position and genomic features, with low sequence homologies to known picornaviruses especially in leader and 2A proteins. Among the viruses that belong to the closely related bat picornavirus groups 1 to 3 and the genus Sapelovirus, G+C content and sequence analysis of P1, P2, and P3 regions showed that FePV is most closely related to bat picornavirus group 3. However, FePV possessed other distinct features, including a putative type IV internal ribosome entry site/segment (IRES) instead of type I IRES in bat picornavirus group 3, protein cleavage sites, and H-D-C catalytic triad in 3Cpro different from those in sapeloviruses and bat picornaviruses, and the shortest leader protein among known picornaviruses. These results suggest that FePV may belong to a new genus in the family Picornaviridae. Western blot analysis using recombinant FePV VP1 polypeptide showed a high seroprevalence of 33.6% for IgG among the plasma samples from 232 cats tested. IgM was also detected in three cats positive for FePV in fecal samples, supporting recent infection in these cats. Further studies are important to understand the pathogenicity, epidemiology, and genetic evolution of FePV in these common pet animals.

Discovery of a novel bottlenose dolphin coronavirus reveals a distinct species of marine mammal coronavirus in Gammacoronavirus

ABSTRACT While gammacoronaviruses mainly comprise infectious bronchitis virus (IBV) and its closely related bird coronaviruses (CoVs), the only mammalian gammacoronavirus was discovered from a white beluga whale (beluga whale CoV [BWCoV] SW1) in 2008. In this study, we discovered a novel gammacoronavirus from fecal samples from three Indo-Pacific bottlenose dolphins (Tursiops aduncus), which we named bottlenose dolphin CoV (BdCoV) HKU22. All the three BdCoV HKU22-positive samples were collected on the same date, suggesting a cluster of infection, with viral loads of 1 × 103 to 1 × 105 copies per ml. Clearance of virus was associated with a specific antibody response against the nucleocapsid of BdCoV HKU22. Complete genome sequencing and comparative genome analysis showed that BdCoV HKU22 and BWCoV SW1 have similar genome characteristics and structures. Their genome size is about 32,000 nucleotides, the largest among all CoVs, as a result of multiple unique open reading frames (NS5a, NS5b, NS5c, NS6, NS7, NS8, NS9, and NS10) between their membrane (M) and nucleocapsid (N) protein genes. Although comparative genome analysis showed that BdCoV HKU22 and BWCoV SW1 should belong to the same species, a major difference was observed in the proteins encoded by their spike (S) genes, which showed only 74.3 to 74.7% amino acid identities. The high ratios of the number of synonymous substitutions per synonymous site (Ks ) to the number of nonsynonymous substitutions per nonsynonymous site (Ka ) in multiple regions of the genome, especially the S gene (Ka /Ks ratio, 2.5), indicated that BdCoV HKU22 may be evolving rapidly, supporting a recent transmission event to the bottlenose dolphins. We propose a distinct species, Cetacean coronavirus, in Gammacoronavirus, to include BdCoV HKU22 and BWCoV SW1, whereas IBV and its closely related bird CoVs represent another species, Avian coronavirus, in Gammacoronavirus.

Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry for identification of clinically significant bacteria that are difficult to identify in clinical laboratories

Aims Although the revolutionary matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) has been evaluated for identification of various groups of bacteria, its application in bacteria that are ‘difficult-to-identify’ by phenotypic tests has been less well studied. We aim to evaluate the usefulness of MALDI-TOF MS for identification of ‘difficult-to-identify’ bacterial isolates. Methods We evaluated the performance of the Bruker MALDI-TOF MS system for a collection of 67 diverse clinically important bacterial isolates that were less commonly encountered, possessed ambiguous biochemical profiles or belonged to newly discovered species. The results were compared with 16S rRNA gene sequencing as a reference method for species identification. Results Using 16S rRNA gene sequencing as the reference method, 30 (45%) isolates were identified correctly to species level (score ≥2.0), 20 (30%) were only identified to genus level (score ≥1.7), four (6%) were misidentified (incorrect species with score ≥2.0 or incorrect genus with score ≥1.7) and 13 (19%) showed ‘no identification’ (score <1.7). Aerobic Gram-positive bacteria showed the highest percentage of correct species identification, followed by aerobic Gram-negative, anaerobic Gram-positive and anaerobic Gram-negative bacteria. Sixteen isolates identified to genus level actually showed the correct species but with scores below the threshold for species identification. Most isolates which showed ‘no identification’ were due to the absence of the corresponding species in the Bruker database. Conclusions Expansion of commercial databases to include reference spectra of less commonly encountered and newly discovered species and to increase available spectra for each species is required to improve the accuracy of MALDI-TOF MS for identifying ‘difficult-to-identify’ bacteria.