Usefulness of Next-Generation Sequencing in Excluding Bovine Leukemia Virus as a Cause of Adult Camel Leukosis in Dromedaries.

Adult camel leukosis is an emerging hematological and neoplastic disease in dromedaries. It has been hypothesized that bovine leukemia virus (BLV) or its genetic variants may be associated with adult camel leukosis. In this study, we used next-generation sequencing (NGS) to detect all possible viruses in five lung samples from five dromedaries with histopathological evidence of adult camel leukosis and four tissue samples from two control dromedaries. A total throughput of 114.7 Gb was achieved, with an average of 12.7 Gb/sample. For each sample, all the pair-end 151-bp reads were filtered to remove rRNA sequences, bacterial genomes and redundant sequences, resulting in 1-7 Gb clean reads, of which <3% matched to viruses. The largest portion of these viral sequences was composed of bacterial phages. About 100-300 reads in each sample matched "multiple sclerosis-associated retrovirus", but manual analysis showed that they were only repetitive sequences commonly present in mammalian genomes. All viral reads were also extracted for analysis, confirming that no BLV or its genetic variants or any other virus was detected in the nine tissue samples. NGS is not only useful for detecting microorganisms associated with infectious diseases, but also important for excluding an infective cause in scenarios where such a possibility is suspected.

Seasonal shift in gut microbiome diversity in wild Sichuan takin (Budorcas tibetanus) and environmental adaptation.

In this study, we investigated the change in microbiome composition of wild Sichuan takin (Budorcas tibetanus) during winter and spring and analyzed the physiological implications for such changes. Diversity analyses of the microbiome (average 15,091 high-quality reads per sample) in 24 fecal samples (15 from winter, 9 from spring) revealed that spring samples had higher species diversity and were compositionally different from winter samples (P < 0.05). Taxonomic composition analysis showed that the relative abundance increased in spring for Patescibacteria (2.7% vs. 0.9% in winter, P < 0.001) and Tenericutes (1.9% vs. 1% in winter, P < 0.05). Substantial increases in relative abundance of Ruminococcaceae and Micrococcaceae were identified in spring and winter, respectively. Mann-Whitney U and ANCOM identified seven differentially abundant genera: Enterococcus, Acetitomaculum, Blautia, Coprococcus 1, Lachnospiraceae UCG 008, Ruminococcus 2 and Ralstonia. All seven genera were significantly more abundant in spring (average 0.016-1.2%) than winter (average 0-0.16%), with the largest difference found in Ruminococcus (1.21% in spring vs. 0.16% in winter). The other six genera were undetectable in winter. Functional prediction and pathway analysis revealed that biosynthesis of cofactors (ko01240) had the highest gene count ratios in the winter, followed by the two-component system (ko02020). Seasonal variation affects the gut microbiomes in wild Sichuan takins, with winter associated with lower species diversity and spring with enrichment of cellulose-degrading genera and phytopathogens. Such changes were crucial in their adaptation to the environment, particularly the difference in food abundance.

Streptococcus oriscaviae sp. nov. Infection Associated with Guinea Pigs.

Pet bite-related infections are commonly caused by the pet's oral flora transmitted to the animal handlers through the bite wounds. In this study, we isolated a streptococcus, HKU75T, in pure culture from the purulent discharge collected from a guinea pig bite wound in a previously healthy young patient. HKU75T was alpha-hemolytic on sheep blood agar and agglutinated with Lancefield group D and group G antisera. API 20 STREP showed that the most likely identity for HKU75T was S. suis I with 85.4% confidence while Vitek 2 showed that HKU75T was unidentifiable. MALDI-TOF MS identified HKU75T as Streptococcus suis (score of 1.86 only). 16S rRNA gene sequencing showed that HKU75T was most closely related to S. parasuis (98.3% nucleotide identity), whereas partial groEL and rpoB gene sequencing showed that it was most closely related to S. suis (81.8% and 89.8% nucleotide identity respectively). Whole genome sequencing and intergenomic distance determined by ANI revealed that there was <85% identity between the genome of HKU75T and those of all other known Streptococcus species. Genome classification using concatenated sequences of 92 bacterial core genes showed that HKU75T belonged to the Suis group. groEL gene sequences identical to that of HKU75T could be directly amplified from the oral cavities of the two guinea pigs owned by the patient. HKU75T is a novel Streptococcus species, which we propose to be named S. oriscaviae. The oral cavity of guinea pigs is presumably a reservoir of S. oriscaviae. Some of the reported S. suis strains isolated from clinical specimens may be S. oriscaviae. IMPORTANCE We reported the discovery of a novel Streptococcus species, propose to be named Streptococcus oriscaviae, from the pus collected from a guinea pig bite wound in a healthy young patient. The bacterium was initially misidentified as S. suis/S. parasuis by biochemical tests, mass spectrometry. and housekeeping genes sequencing. Its novelty was confirmed by whole genome sequencing. Comparative genomic studies showed that S. oriscaviae belongs to the Suis group. S. oriscaviae sequences were detected in the oral cavities of the two guinea pigs owned by the patient, suggesting that the oral cavity of guinea pigs could be a reservoir of S. oriscaviae. Some of the reported S. suis strains may be S. oriscaviae. Further studies are warranted to refine our knowledge on this novel Streptococcus species.

Substantial Decline in Invasive Pneumococcal Disease During Coronavirus Disease 2019 Pandemic in Hong Kong.

Compared with other countries, a more substantial decrease in the incidence of invasive pneumococcal disease was observed in Hong Kong, which is most likely attributable to the proactive mass adoption of face masks by the public. Human behavioral changes, particularly mask wearing, should be considered as an additional preventive strategy against invasive pneumococcal disease.

In Vitro Susceptibility of Typhoidal, Nontyphoidal, and Extended-Spectrum-ß-Lactamase-Producing Salmonella to Ceftolozane/Tazobactam.

Both typhoidal and nontyphoidal salmonellae are included in the top 15 drug-resistant threats described by the U.S. Centers for Disease Control and Prevention. There is an urgent need to look for alternative antibiotics for the treatment of Salmonella infections. We used the broth microdilution test to examine the in vitro susceptibilities of typhoidal and nontyphoidal salmonellae, including isolates positive for extended-spectrum ß-lactamase (ESBL), to ceftolozane/tazobactam and six other antibiotics. Of the 313 (52 typhoidal and 261 nontyphoidal) Salmonella isolates tested, 98.7% were susceptible to ceftolozane/tazobactam. Based on the overall MIC50/90 values, Salmonella isolates were more susceptible to ceftolozane/tazobactam (0.25/0.5 mg/L) than all the comparator agents: ampicillin (≥64/≥64 mg/L), levofloxacin (0.25/1 mg/L), azithromycin (4/16 mg/L), ceftriaxone (≤0.25/4 mg/L), chloramphenicol (8/≥64 mg/L), and trimethoprim/sulfamethoxazole (1/≥8 mg/L). Comparison of the activities of the antimicrobial agents against nontyphoidal Salmonella isolates according to their serogroups showed that ceftolozane/tazobactam had the highest activity (100%) against Salmonella serogroup D, G, I, and Q isolates, whereas the lowest activity (85.7%) was observed against serogroup E isolates. All 10 ESBL-producing Salmonella isolates (all nontyphoidal), of which 8 were CTX-M-55 producers and 2 were CTX-M-65 producers, were sensitive to ceftolozane/tazobactam, albeit with MIC50/90 values higher (1/2 mg/L) than those for non-ESBL producers (0.25/0.5 mg/L). In summary, our data indicate that ceftolozane/tazobactam is active against most strains of both typhoidal and nontyphoidal salmonellae and also against ESBL-producing salmonellae.

Development of a sensitive competitive enzyme-linked immunosorbent assay for serodiagnosis of Burkholderia mallei, a Tier 1 select agent.

Glanders is a highly contagious and potentially serious disease caused by Burkholderia mallei, a Tier 1 select agent. In this study, we raised a monoclonal antibody (mAb) against the lipopolysaccharide (LPS) of B. mallei and developed a competitive enzyme-linked immunosorbent assay (cELISA) for B. mallei infection. Using the titrated optimal conditions of B. mallei-LPS (2 ng) for microtiter plate coating, sample serum dilution at 1:20 and 3.5 ng/µL anti-LPS mAb B5, the cutoff value of the cELISA was determined using serum samples from 136 glanders-free seronegative horses in Hong Kong. All calculated percentage inhibition (PI) values from these seronegative samples were below 39.6% inhibition (1.5 standard deviations above mean PI) and was used as the cutoff value. The diagnostic sensitivity of the developed LPS-based cELISA was first evaluated using sera from donkeys and mice inoculated with B. mallei. An increasing trend of PI values above the defined cELISA cutoff observed in the donkey and mouse sera suggested positive detection of anti-LPS antibodies. The sensitivity and specificity of the LPS-based cELISA was further evaluated using 31 serologically positive horse sera from glanders outbreaks in Bahrain and Kuwait, of which 30 were tested positive by the cELISA; and 21 seronegative horse sera and 20 seronegative donkey sera from Dubai, of which all were tested negative by the cELISA. A cELISA with high sensitivity (97.2%) and specificity (100%) for the detection of B. mallei antibodies in different animals was developed.

Rapid Genomic Diagnosis of Fungal Infections in the Age of Next-Generation Sequencing.

Next-generation sequencing (NGS) technologies have recently developed beyond the research realm and started to mature into clinical applications. Here, we review the current use of NGS for laboratory diagnosis of fungal infections. Since the first reported case in 2014, >300 cases of fungal infections diagnosed by NGS were described. Pneumocystis jirovecii is the predominant fungus reported, constituting ~25% of the fungi detected. In ~12.5% of the cases, more than one fungus was detected by NGS. For P. jirovecii infections diagnosed by NGS, all 91 patients suffered from pneumonia and only 1 was HIV-positive. This is very different from the general epidemiology of P. jirovecii infections, of which HIV infection is the most important risk factor. The epidemiology of Talaromyces marneffei infection diagnosed by NGS is also different from its general epidemiology, in that only 3/11 patients were HIV-positive. The major advantage of using NGS for laboratory diagnosis is that it can pick up all pathogens, particularly when initial microbiological investigations are unfruitful. When the cost of NGS is further reduced, expertise more widely available and other obstacles overcome, NGS would be a useful tool for laboratory diagnosis of fungal infections, particularly for difficult-to-grow fungi and cases with low fungal loads.

A Sensitive and Specific Competitive Enzyme-Linked Immunosorbent Assay for Serodiagnosis of COVID-19 in Animals.

In addition to human cases, cases of COVID-19 in captive animals and pets are increasingly reported. This raises the concern for two-way COVID-19 transmission between humans and animals. Here, we developed a SARS-CoV-2 nucleocapsid protein-based competitive enzyme-linked immunosorbent assay (cELISA) for serodiagnosis of COVID-19 which can theoretically be used in virtually all kinds of animals. We used 187 serum samples from patients with/without COVID-19, laboratory animals immunized with inactive SARS-CoV-2 virions, COVID-19-negative animals, and animals seropositive to other betacoronaviruses. A cut-off percent inhibition value of 22.345% was determined and the analytical sensitivity and specificity were found to be 1:64-1:256 and 93.9%, respectively. Evaluation on its diagnostic performance using 155 serum samples from COVID-19-negative animals and COVID-19 human patients showed a diagnostic sensitivity and specificity of 80.8% and 100%, respectively. The cELISA can be incorporated into routine blood testing of farmed/captive animals for COVID-19 surveillance.

High Prevalence of Genogroup I and Genogroup II Picobirnaviruses in Dromedary Camels.

Picobirnaviruses (PBVs) are small non-enveloped bisegmented double-stranded RNA viruses found in humans, mammals, and birds. Increasing molecular epidemiology studies suggest a high sequence diversity of PBVs in numerous hosts and the environment. In this study, using 229 fecal samples from dromedary camels in Dubai, 52.8% were positive for PBVs, of which 77.7% and 41.3% were positive for genogroup I and II, respectively, and 19.0% were positive for both genotypes. Phylogenetic analysis showed high diversity among the sequences of genogroup I and II dromedary PBVs. Marked nucleotide polymorphisms were observed in 75.5% and 46.0% of genogroup I and II RNA-dependent RNA polymerase (RdRp) sequences, respectively, suggesting the co-existence of multiple strains in the same specimen. Both high genetic diversity and prevalence of genogroup I and II PBV in dromedaries were observed. In fact, the prevalence of genogroup II PBV in dromedaries is the highest among all animals to date. The complete/near-complete core genomes of five genogroup I and one genogroup II dromedary PBVs and partial segment 1 and 2 of both genotypes were also sequenced. The dromedary PBV genome organizations were similar to those of other animals. Genetic reassortment and mutation are both important in the ecology and evolution of PBVs.

High Prevalence and Mechanism Associated With Extended Spectrum Beta-Lactamase-Positive Phenotype in Laribacter hongkongensis.

In this study, we reported the prevalence and mechanism associated with the extended-spectrum beta-lactamase (ESBL)-positive phenotype in Laribacter hongkongensis isolated from patients and fish. Using the inhibition zone enhancement test, 20 (95.2%) of the 21 patient strains and 8 (57.1%) of the 14 fish strains were tested ESBL-positive. However, ESBL genes, including SHV, TEM, CTX-M, GES, and PER, were not detected in all of these 28 L. hongkongensis isolates. No ESBL gene could be detected in either the complete genome of L. hongkongensis HLHK9 or the draft genome of PW3643. PCR and DNA sequencing revealed that all the 35 L. hongkongensis isolates (showing both ESBL-positive and ESBL-negative phenotypes) were positive for the ampC gene. When the AmpC deletion mutant, HLHK9ΔampC, was subject to the zone enhancement test, the difference of zone size between ceftazidime/clavulanate and ceftazidime was less than 5 mm. When boronic acid was added to the antibiotic disks, none of the 28 "ESBL-positive" isolates showed a ≥ 5 mm enhancement of inhibition zone size diameter between ceftazidime/clavulanate and ceftazidime and between cefotaxime/clavulanate and cefotaxime. A high prevalence (80%) of ESBL-positive phenotype is present in L. hongkongensis. Overall, our results suggested that the ESBL-positive phenotype in L. hongkongensis results from the expression of the intrinsic AmpC beta-lactamase. Confirmatory tests should be performed before issuing laboratory reports for L. hongkongensis isolates that are tested ESBL-positive by disk diffusion clavulanate inhibition test.

Middle East Respiratory Syndrome Coronavirus Antibodies in Bactrian and Hybrid Camels from Dubai.

So far, dromedary camels are the only known animal reservoir for Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV). Previous published serological studies showed that sera of Bactrian camels were all negative for MERS-CoV antibodies. However, a recent study revealed that direct inoculation of Bactrian camels intranasally with MERS-CoV can lead to infection with abundant virus shedding and seroconversion. In this study, we examined the presence of MERS-CoV antibodies in Bactrian and hybrid camels in Dubai, the United Arab Emirates (where dromedaries are also present), and Bactrian camels in Xinjiang, China (where dromedaries are absent). For the 29 serum samples from Bactrian camels in Dubai tested by the MERS-CoV spike (S) protein-based enzyme-linked immunosorbent assay (S-ELISA) and neutralization antibody test, 14 (48%) and 12 (41%), respectively, were positive for MERS-CoV antibodies. All the 12 serum samples that were positive with the neutralization antibody test were also positive for the S-ELISA. For the 11 sera from hybrid camels in Dubai tested with the S-ELISA and neutralization antibody test, 6 (55%) and 9 (82%), respectively, were positive for MERS-CoV antibodies. All the 6 serum samples that were positive for the S-ELISA were also positive with the neutralization antibody test. There was a strong correlation between the antibody levels detected by S-ELISA and neutralizing antibody titers, with a Spearman coefficient of 0.6262 (P < 0.0001; 95% confidence interval, 0.5062 to 0.7225). All 92 Bactrian camel serum samples from Xinjiang were negative for MERS-CoV antibodies tested using both S-ELISA and the neutralization antibody test. Bactrian and hybrid camels are potential sources of MERS-CoV infection.IMPORTANCE Since its first appearance in 2012, Middle East respiratory syndrome (MERS) has affected >25 countries, with >2,400 cases and an extremely high fatality rate of >30%. The total number of mortalities due to MERS is already greater than that due to severe acute respiratory syndrome. MERS coronavirus (MERS-CoV) has been confirmed to be the etiological agent. So far, dromedaries are the only known animal reservoir for MERS-CoV. Previously published serological studies showed that sera of Bactrian camels were all negative for MERS-CoV antibodies. In this study, we observed that 41% of the Bactrian camel sera and 55% of the hybrid camel sera from Dubai (where dromedaries are also present), but none of the sera from Bactrian camels in Xinjiang (where dromedaries are absent), were positive for MERS-CoV antibodies. Based on these results, we conclude that in addition to dromedaries, Bactrian and hybrid camels are also potential sources of MERS-CoV infection.

Severe underlying liver diseases and high mortality associated with Laribacter hongkongensis bacteremia.

We characterized a strain of Laribacter hongkongensis isolated from the blood of a patient with fatal sepsis, who had alcoholic cirrhosis with ascites and portal hypertension. L. hongkongensis bacteremia is associated with underlying liver diseases (P < 0.001) and mortality (P < 0.05), whereas L. hongkongensis gastroenteritis is associated with recent travel history (P < 0.05).

Tsukamurella asaccharolytica sp. nov., Tsukamurella conjunctivitidis sp. nov. and Tsukamurella sputi sp. nov., isolated from patients with bacteraemia, conjunctivitis and respiratory infection in Hong Kong.

Three bacterial strains, HKU70T, HKU71T and HKU72T, were isolated from the conjunctival swab, blood and sputum samples of three patients with conjunctivitis, bacteraemia and respiratory infection, respectively, in Hong Kong. The three strains were aerobic, Gram-stain positive, catalase-positive, non-sporulating and non-motile bacilli and exhibited unique biochemical profiles distinguishable from currently recognized Tsukamurella species. 16S rRNA, secA, rpoB and groEL gene sequence analyses revealed that the three strains shared 99.6-99.9, 94.5-96.8, 95.7-97.8 and 97.7-98.9 % nucleotide identities with their corresponding closest Tsukamurella species respectively. DNA-DNA hybridization confirmed that they were distinct from other known species of the genus Tsukamurella (26.2±2.4 to 36.8±1.2 % DNA-DNA relatedness), in line with results of in silico genome-to-genome comparison (32.2-40.9 % Genome-to-Genome Distance Calculator and 86.3-88.9 % average nucleotide identity values]. Fatty acids, mycolic acids, cell-wall sugars and peptidoglycan analyses showed that they were typical of members of Tsukamurella. The G+C content determined based on the genome sequence of strains HKU70T, HKU71T and HKU72T were 69.9, 70.2 and 70.5 mol%, respectively. Taken together, our results supported the proposition and description of three new species, i.e. Tsukamurella sputi HKU70T (=JCM 33387T=DSM 109106T) sp. nov., Tsukamurella asaccharolytica HKU71T (=JCM 33388T=DSM 109107T) sp. nov. and Tsukamurella conjunctivitidis HKU72T (=JCM 33389T=DSM 109108T) sp. nov.

Outer membrane protein A (OmpA) is a potential virulence factor of Vibrio alginolyticus strains isolated from diseased fish.

Vibrio alginolyticus is one of the most serious causative agents of diseases in cultured marine fish and shellfish. However, the characteristics of virulence factors in pathogenic V. alginolyticus are poorly known. To gain insight into fish diseases caused by V. alginolyticus, we carried out two-dimensional gel electrophoresis (2-DE) combined with MALDI-TOF mass spectrometry to identify uniquely expressed proteins in the disease-causing V. alginolyticus. V. alginolyticus strains were isolated from marine environments and diseased fish obtained from southern Thailand. We identified seven unique proteins in the disease-causing V. alginolyticus strain. Among those, the outer membrane protein A (OmpA) had the strongest expression. Therefore, the function of this protein was further analysed. To investigate the role of OmpA protein, an in-frame deletion mutant of ompA was constructed using the homologous recombination method. Although the ompA mutant V. alginolyticus strain (ΔompA) grew normally, the mutant exhibited a significant defect in the swarming ability and the biofilm formation. Furthermore, Galleria mellonella larvae injected with the mutant bacteria had a significantly greater survival percentage than those injected with the wild-type strain, demonstrating that OmpA protein is required for the pathogenicity of V. alginolyticus. Together, this study suggests a potential target for vaccine development against pathogenic V. alginolyticus strain.

Influenza A(H1N1)pdm09 Virus Infection in a Captive Giant Panda, Hong Kong.

We report influenza A(H1N1)pdm09 virus infection in a captive giant panda in Hong Kong. The viral load peaked on day 1 and became undetectable on day 5, and an antibody response developed. Genome analysis showed 99.3%-99.9% nucleotide identity between the virus and influenza A(H1N1)pdm09 virus circulating in Hong Kong.

Malate-Dependent Carbon Utilization Enhances Central Metabolism and Contributes to Biological Fitness of Laribacter hongkongensis via CRP Regulation.

Metabolic adaptation in various environmental niches is crucial for bacterial extracellular survival and intracellular replication during infection. However, the metabolism of carbon/nitrogen sources and related regulatory mechanisms in Laribacter hongkongensis, an asaccharolytic bacterium associated with invasive infections and gastroenteritis, are still unknown. In the present study, we demonstrated that malate can be exploited as a preferred carbon source of L. hongkongensis. Using RNA-sequencing, we compared the transcription profiles of L. hongkongensis cultivated with or without malate supplementation, and observed that malate utilization significantly inhibits the use of alternative carbon sources while enhancing respiratory chain as well as central carbon, sulfur, and urease-mediated nitrogen metabolisms. The tight connection among these important metabolic pathways indicates that L. hongkongensis is capable of integrating information from different metabolism branches to coordinate the expression of metabolic genes and thereby adapt to environmental changing. Furthermore, we identified that a transcription factor, CRP, is repressed by malate-mediated metabolism while negatively regulating the effect of malate on these central metabolic pathways. Remarkably, CRP also responds to various environmental stresses, influences the expression of other transcription factors, and contributes to the biological fitness of L. hongkongensis. The regulatory network and cross-regulation enables the bacteria to make the appropriate metabolic responses and environmental adaptation.

Novel Picobirnaviruses in Respiratory and Alimentary Tracts of Cattle and Monkeys with Large Intra- and Inter-Host Diversity.

Picobirnaviruses (PBVs) are mostly found in animal alimentary samples. In this study, among 576 respiratory specimens from 476 mammals and 100 chickens, genogroup I PBVs were detected in three cattle and three monkeys, and a genogroup II PBV-positive sample was collected from one cattle specimen. More than one PBV sequence type was observed in two and one genogroup I PBV-positive samples from cattle and monkeys, respectively. Twenty-four complete/near-complete segments 2 (nine from respiratory and 15 from alimentary samples) from the cattle and monkey genogroup I PBVs and one complete segment 2 from the cattle genogroup II PBV were sequenced. Similar to other studies, the cattle PBVs also showed a high diversity. In contrast, the monkey PBVs observed in this study were clustered into three distinct clades. Within each clade, all the sequences showed >99% amino acid identities. This unique phenomenon is probably due to the fact that monkeys in our locality reside in separated troops with minimal inter-troop contact.

Novel Bat Alphacoronaviruses in Southern China Support Chinese Horseshoe Bats as an Important Reservoir for Potential Novel Coronaviruses.

While bats are increasingly recognized as a source of coronavirus epidemics, the diversity and emergence potential of bat coronaviruses remains to be fully understood. Among 1779 bat samples collected in China, diverse coronaviruses were detected in 32 samples from five different bat species by RT-PCR. Two novel alphacoronaviruses, Rhinolophus sinicus bat coronavirus HKU32 (Rs-BatCoV HKU32) and Tylonycteris robustula bat coronavirus HKU33 (Tr-BatCoV HKU33), were discovered from Chinese horseshoe bats in Hong Kong and greater bamboo bats in Guizhou Province, respectively. Genome analyses showed that Rs-BatCoV HKU32 is closely related to BatCoV HKU10 and related viruses from diverse bat families, whereas Tr-BatCoV HKU33 is closely related to BtNv-AlphaCoV and similar viruses exclusively from bats of Vespertilionidae family. The close relatedness of Rs-BatCoV HKU32 to BatCoV HKU10 which was also detected in Pomona roundleaf bats from the same country park suggests that these viruses may have the tendency of infecting genetically distant bat populations of close geographical proximity with subsequent genetic divergence. Moreover, the presence of SARSr-CoV ORF7a-like protein in Rs-BatCoV HKU32 suggests a common evolutionary origin of this accessory protein with SARS-CoV, also from Chinese horseshoe bats, an apparent reservoir for coronavirus epidemics. The emergence potential of Rs-BatCoV HKU32 should be explored.

Transmission of a Novel Genotype of Hepatitis E Virus from Bactrian Camels to Cynomolgus Macaques.

Hepatitis E virus (HEV) is zoonotic and a major cause of acute viral hepatitis worldwide. Recently, we identified a novel HEV genotype 8 (HEV8) in Bactrian camels in Xinjiang, China. However, the epidemiology, pathogenicity, and zoonotic potential of HEV8 are unclear. Here, we present the prevalence of HEV8 in China and investigate its pathogenicity and cross-species transmission in cynomolgus macaques. Fresh fecal and milk samples from Bactrian camels collected from four provinces/regions in China were screened for HEV RNA by reverse transcriptase PCR (RT-PCR). An HEV8-positive sample was used to inoculate two cynomolgus macaques to examine the potential for cross-species infection. The pathogenicity of HEV8 was analyzed by testing HEV markers and liver function during the study period and histopathology of liver biopsy specimens at 3, 13, and 25 weeks postinoculation. Extrahepatic replication was tested by using reverse transcriptase quantitative PCR (RT-qPCR) and immunofluorescence assays. The overall prevalence of HEV8 RNA in Chinese Bactrian camels was 1.4% (4/295), and positive samples were found in three different provinces/regions in China. Histopathology confirmed acute and chronic HEV8 infections in the two monkeys. Multiple tissues were positive for HEV RNA and ORF2 proteins. Renal pathology was observed in the monkey with chronic hepatitis. Whole-genome sequencing showed only 1 to 3 mutations in the HEV8 in the fecal samples from the two monkeys compared to that from the camel. HEV8 is circulating in multiple regions in China. Infection of two monkeys with HEV8 induced chronic and systemic infections, demonstrating the high potential zoonotic risk of HEV8.IMPORTANCE It is estimated that one-third of the world population have been exposed to hepatitis E virus (HEV). In developed countries and China, zoonotic HEV strains are responsible for almost all acute and chronic HEV infection cases. It is always of immediate interest to investigate the zoonotic potential of novel HEV strains. In 2016, we discovered a novel HEV genotype, HEV8, in Bactrian camels, but the epidemiology, zoonotic potential, and pathogenicity of the virus were unknown. In the present study, we demonstrated that HEV8 was circulating in multiple regions in China and was capable of infecting cynomolgus macaques, a surrogate for humans, posing high risk of zoonosis. Chronic hepatitis, systemic infection, and renal pathology were observed. Collectively, these data indicate that HEV8 exhibits a high potential for zoonotic transmission. Considering the importance of Bactrian camels as livestock animals, risk groups, such as camelid meat and milk consumers, should be screened for HEV8 infection.

First case report of fatal Nocardia nova infection in yellow-bibbed lory (Lorius chlorocercus) identified by multilocus sequencing.

BACKGROUND: Nocardiosis is often a multi-systemic disease in humans and other mammals. Nocardiosis in birds is uncommon. Laboratory identification of Nocardia to the species level is difficult by traditional phenotypic methods based on biochemical reactions and hydrolysis tests, and is most accurately performed by sequencing multiple gene targets. CASE PRESENTATION: We report the first case of fatal Nocardia nova infection in a yellow-bibbed lory nestling in an oceanarium diagnosed by multilocus sequencing. Necropsy examination showed effacement of normal sternal musculature with yellowish, firm aberrant material, and diffuse infiltration of the lungs with nodular, tan to yellow foci. Histologically, severe granulomatous inflammation with marked necrosis was observed in the lung, spleen and sternal musculature. Fine, sometimes Gram-positive, 0.5-1 µm wide, branching and beaded filamentous organisms were visible within the lesions. They were acid-fast on Fite-Faraco stain. Tissue samples obtained from the sternum, liver, right lung and right kidney recovered Nocardia species. Sequencing of four gene loci and phylogenetic analysis of concatenated (gyrB-16S-secA1-hsp65) sequences revealed that the isolate was N. nova. CONCLUSIONS: We report the first case of N. nova infection in yellow-bibbed lorry (Lorius chlorocercus). The present case is the first one of which the species identity of the isolate was determined by multilocus sequencing. Molecular diagnosis is important for identifying the Nocardia to species level and understanding the epidemiology of nocardiosis in birds.