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Due to the increasing resistance of aerobic and facultative anaerobic Gram-negative rods, ceftazidime-avibactam and ceftolozane-tazobactam have been launched in the market in the last few years. In this study, we analyzed the susceptibility pattern of the major aerobic and facultative anaerobic Gram-negative rods in Hong Kong for ceftazidime-avibactam, ceftolozane-tazobactam, four other broad-spectrum antibiotics commonly used in Hong Kong and colistin. For 300 isolates collected from January to December 2021, non-ESBL-producing Enterobacterales, ESBL-producing Enterobacterales and Pseudomonas aeruginosa were highly susceptible to ceftazidime-avibactam (all 100%) and ceftolozane-tazobactam (98.7%, 99.7% and 94.3%). For 32 archived ESBL-producing Klebsiella pneumoniae isolates collected between January 2014 and March 2023, all were susceptible to ceftazidime-avibactam and ceftolozane-tazobactam. For 101 archived carbapenemase-producing Enterobacterales, their susceptibilities to ceftazidime-avibactam and ceftolozane-tazobactam varied depending on the type of carbapenemase produced. Both had high activities against OXA-producing strains (97.1% and 76.5%, respectively) but were 100% resistant for NDM-producing and NDM+OXA-producing strains. All KPC-producing strains were susceptible to ceftazidime-avibactam but resistant to ceftolozane-tazobactam. Ceftazidime-avibactam and ceftolozane-tazobactam are good alternatives for the management of infections caused by ESBL-producing Enterobacterales and selective strains of carbapenemase-producing Enterobacterales in Hong Kong.

ABSTRACT Talaromyces marneffei is a thermally dimorphic fungal pathogen endemic in Southeast Asia. As inhalation of airborne conidia is believed as the major infection route, airway epithelial cells followed by pulmonary macrophages are the first cell types which the fungus encounters inside the host. In this study, we established an in vitro infection model based on human peripheral blood-derived macrophages (hPBDMs) cultured with the supplementation of autologous plasma. Using this model, we determined the transcriptomic changes of hPBDMs in response to T. marneffei infection by quantitative real-time reverse-transcription polymerase chain reaction as well as high-throughput RNA sequencing. Results showed that T. marneffei infection could activate hPBDMs to the M1-like phenotype and trigger a potent induction of chemokine and pro-inflammatory cytokine production as well as the expression of other immunoregulatory genes. In contrast to hPBDMs, there was no detectable innate cytokine response against T. marneffei in human bronchial epithelial cells (hBECs). Using a green fluorescent protein-tagged T. marneffei strain and confocal microscopy, internalization of the fungus by hBECs was confirmed. Live cell imaging further demonstrated that the infected cells exhibited normal cellular physiology, especially that the process of cell division could be observed. Moreover, T. marneffei also survived better inside hBECs than hPBDMs. Our results illustrated a potential role of hBECs to serve as reservoir cells for T. marneffei to evade immunosurveillance by phagocytes, from which the fungus reactivates when the host immunity is weakened and causes infection. Such immunoevasion and reactivation may also help explain the long incubation period observed for talaromycosis, in particular the travel-related cases. IMPORTANCE Talaromyces marneffei is an important fungal pathogen especially in Southeast Asia. To understand the innate immune response to talaromycosis, a suitable infection model is needed. Here, we established an in vitro T. marneffei infection model using human peripheral blood-derived macrophages (hPBDMs). We then examined the transcriptomic changes of hPBDMs in response to T. marneffei infection with this model. We found that contact with T. marneffei could activate hPBDMs to the M1-like phenotype and induced mRNA expressions of five cytokines and eight immunoregulatory genes. Contrary to hPBDMs, such immunoresponse was not elicited in human bronchial epithelial cells (hBECs), despite normal physiology observed in infected cells. We also found that infected hBECs did not eliminate T. marneffei as efficiently as hPBDMs. Our observation suggested that hBECs may potentially serve as reservoir cells for T. marneffei to evade immunosurveillance. When the host immunity deteriorates later, then the fungus reactivates and causes infection.

Nocardia species do not replicate as rapidly as other pyogenic bacteria and nocardial infections can be highly fatal, particularly in immunocompromised patients. Here, we present the first report of fatal Nocardia kroppenstedtii bacteremic pneumonia and empyema thoracis diagnosed by next-generation sequencing (NGS) using the Oxford Nanopore Technologies' MinION device. The bacterium was not identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Due to its low equipment cost, short turn-around-time, and portable size, the Oxford Nanopore Technologies' MinION device is a useful platform for NGS in routine clinical microbiology laboratories.

Irrespective of whether COVID-19 originated from a natural or a genetically engineered virus, the ultimate source of Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is bats [...]

Abstract. Although various opportunistic infections have been described in patients with anti-interferon gamma autoantibodies, so far there is no Listeria monocytogenes infection reported to be associated with this primary immunodeficiency. Here, we describe the first case of bacteremic L. monocytogenes meningitis in a 59-year-old Chinese man with anti-interferon gamma autoantibodies, who presented with acute onset of fever and severe headache. Blood culture was positive but culture of the cerebrospinal fluid was negative, although it showed features suggestive of partially treated bacterial meningitis. The presence of L. monocytogenes in the cerebrospinal fluid was confirmed by next-generation sequencing. Avoidance of high-risk food items in these patients is important for the prevention of listeriosis. The use of antibiotic regimens that cover Listeria is crucial for empirical treatment, particularly if such patients develop acute or subacute meningitis. Next-generation sequencing is becoming an important diagnostic modality for culture-negative infections.

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

The COVID-19 pandemic response has shown how vaccine platform technologies can be used to rapidly and effectively counteract a novel emerging infectious disease. The speed of development for mRNA and vector-based vaccines outpaced those of subunit vaccines, however, subunit vaccines can offer advantages in terms of safety and stability. Here we describe a subunit vaccine platform technology, the molecular clamp, in application to four viruses from divergent taxonomic families: Middle Eastern respiratory syndrome coronavirus (MERS-CoV), Ebola virus (EBOV), Lassa virus (LASV) and Nipah virus (NiV). The clamp streamlines subunit antigen production by both stabilising the immunologically important prefusion epitopes of trimeric viral fusion proteins while enabling purification without target-specific reagents by acting as an affinity tag. Conformations for each viral antigen were confirmed by monoclonal antibody binding, size exclusion chromatography and electron microscopy. Notably, all four antigens tested remained stable over four weeks of incubation at 40°C. Of the four vaccines tested, a neutralising immune response was stimulated by clamp stabilised MERS-CoV spike, EBOV glycoprotein and NiV fusion protein. Only the clamp stabilised LASV glycoprotein precursor failed to elicit virus neutralising antibodies. MERS-CoV and EBOV vaccine candidates were both tested in animal models and found to provide protection against viral challenge.

ABSTRACT 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

Several human coronaviruses (CoV) have been proposed to emerge from bats but evidence of direct bat-to-human transmission is slim. In this work, the authors isolate a MERS-related CoV strain directly from bats and show that it infects target cells in vitro and engineered mice through the human DDP4 receptor.

Pleurostoma species are wood-inhabiting fungi and emerging opportunistic pathogens causing phaeohyphomycosis. In this study, we isolated a dematiaceous fungus, HKU44T, from the subhepatic abscess pus and drain fluids of a liver transplant recipient with post-transplant biliary and hepatico-jejunostomy bypass strictures. Histology of the abscess wall biopsy showed abundant fungal hyphae. The patient survived after a second liver transplant and antifungal therapy. On SDA, HKU44T grew initially as white powdery colonies which turned beige upon maturation. Hyphae were septate and hyaline. Phialides were monophialidic and laterally located, generally closely associated to a cluster of conidia which were usually reniform. Phylogenetic analyses showed that HKU44T is most closely related to, but distinct from, Pleurostoma ootheca and Pleurostoma repens. These suggested that HKU44T is a novel Pleurostoma species, for which the name Pleurostoma hongkongense sp. nov. is proposed. Antifungal susceptibility testing showed that Pleurostoma species possessed high MICs/MECs for fluconazole, 5-flucytosine and the echinocandins; whereas they exhibited a high strain-to-strain variability to the susceptibilities to the other triazoles. As for amphotericin B, ∼65% of the Pleurostoma strains had low MICs (≤1 µg/mL). DNA sequencing should be performed to accurately identify fungi with Pleurostoma/Phialophora-like morphologies, so is antifungal susceptibility testing for patients with Pleurostoma infections.

Targeted therapies are widely used for treatment of autoimmune diseases as well as solid organ and hematological malignancies. Various opportunistic infections have been described in patients on targeted therapies. Although case reports or a few case series of listeriosis have been reported to be associated with targeted therapy, most of the cases were related to anti-tumor necrosis factor-α monoclonal antibody. In this study, we describe the epidemiological and clinical profiles of listeriosis in a tertiary hospital in Shenzhen, a Southern Chinese metropolitan city in China. During the 9-year-and-6-month study period, a total of five cases of listeriosis were recorded and all of them had Listeria monocytogenes bacteremia. All five patients had predisposing factors, including corticosteroid (n = 3), targeted therapy (n = 2), pregnancy (n = 2) and anti-interferon gamma autoantibody (n = 1). The two patients who had targeted therapy during their course of cancer treatment received inhibitors of the epidermal growth factor receptor (EGFR)/human epidermal growth factor receptor 2 (HER2) pathway. The first one was a 52-year-old woman with metastatic adenocarcinoma of the lung. She was given gefitinib (EGFR tyrosine kinase inhibitor), osimertinib (third-generation EGFR tyrosine kinase inhibitor) and afatinib (tyrosine kinase inhibitor that can bind to EGFR, HER2 and HER4). The second one was a 40-year-old woman with carcinoma of the breast with brain metastasis. She was given trastuzumab (anti-HER2 monoclonal antibody) and lapatinib (dual tyrosine kinase inhibitor of the EGFR/HER2 pathway). These two patients represent the second and third reports of listeria infections associated with EGFR/HER2 pathway inhibitors in the literature. Targeted therapy is an important predisposing factor for listeriosis. Listeria infection is an important differential diagnosis in patients on targeted therapy who present with sepsis and/or central nervous system infection, and the use of antibiotic regimens that cover listeria is crucial for empirical treatment. Avoidance of high-risk food items in these patients is important for the prevention of listeriosis.

We present the first report of histology- and culture-proven Mycobacterium marinum infection diagnosed by next-generation sequencing (NGS). It took

ABSTRACTOnychomycosis is most commonly caused by dermatophytes. In this study, we examined the spectrum of phenotypically non-dermatophyte and non-Aspergillus fungal isolates recovered over a 10-year period from nails of patients with onychomycosis in Hong Kong. A total of 24 non-duplicated isolates recovered from 24 patients were included. The median age of the patients was 51 years, and two-thirds of them were males. One-third and two-thirds had finger and toe nail infections respectively. Among these 24 nail isolates, 17 were confidently identified as 13 different known fungal species, using a polyphasic approach. These 13 species belonged to 11 genera and ≥9 families. For the remaining seven isolates, multilocus sequencing did not reveal their definite species identities. These seven potentially novel species belonged to four different known and three potentially novel genera of seven families. 33.3%, 41.7% and 95.8% of the 24 fungal isolates possessed minimum inhibitory concentrations of >1 µg/mL to terbinafine, itraconazole and fluconazole, respectively, the first line treatment of onychomycosis. A high diversity of moulds was associated with onychomycosis. A significant proportion of the isolates were potentially novel fungal species. To guide proper treatment, molecular identification and antifungal susceptibility testing should be performed for these uncommonly isolated fungal species.

Abstract 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.

Abstract Previous findings of Middle East Respiratory Syndrome coronavirus (MERS-CoV)-related viruses in bats, and the ability of Tylonycteris-BatCoV HKU4 spike protein to utilize MERS-CoV receptor, human dipeptidyl peptidase 4 hDPP4, suggest a bat ancestral origin of MERS-CoV. We developed 12 primary bat cell lines from seven bat species, including Tylonycteris pachypus, Pipistrellus abramus and Rhinolophus sinicus (hosts of Tylonycteris-BatCoV HKU4, Pipistrellus-BatCoV HKU5, and SARS-related-CoV respectively), and tested their susceptibilities to MERS-CoVs, SARS-CoV, and human coronavirus 229E (HCoV-229E). Five cell lines, including P. abramus and R. sinicus but not T. pachypus cells, were susceptible to human MERS-CoV EMC/2012. However, three tested camel MERS-CoV strains showed different infectivities, with only two strains capable of infecting three and one cell lines respectively. SARS-CoV can only replicate in R. sinicus cells, while HCoV-229E cannot replicate in any bat cells. Bat dipeptidyl peptidase 4 (DPP4) sequences were closely related to those of human and non-human primates but distinct from dromedary DPP4 sequence. Critical residues for binding to MERS-CoV spike protein were mostly conserved in bat DPP4. DPP4 was expressed in the five bat cells susceptible to MERS-CoV, with significantly higher mRNA expression levels than those in non-susceptible cells (P = 0.0174), supporting that DPP4 expression is critical for MERS-CoV infection in bats. However, overexpression of T. pachypus DPP4 failed to confer MERS-CoV susceptibility in T. pachypus cells, suggesting other cellular factors in determining viral replication. The broad cellular tropism of MERS-CoV should prompt further exploration of host diversity of related viruses to identify its ancestral origin.

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.

Since its first appearance in the 1960s, the genetic diversity and evolution of human coronavirus 229E (HCoV-229E) have been relatively understudied. In this study, we report a fatal case of COVID-19 coinfected with HCoV-229E in Hong Kong.

ABSTRACT 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 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.

In the last two decades, several coronavirus (CoV) interspecies jumping events have occurred between bats and other animals/humans, leading to major epidemics/pandemics and high fatalities. The SARS epidemic in 2002/2003 had a ~10% fatality. The discovery of SARS-related CoVs in horseshoe bats and civets and genomic studies have confirmed bat-to-civet-to-human transmission. The MERS epidemic that emerged in 2012 had a ~35% mortality, with dromedaries as the reservoir. Although CoVs with the same genome organization (e.g., Tylonycteris BatCoV HKU4 and Pipistrellus BatCoV HKU5) were also detected in bats, there is still a phylogenetic gap between these bat CoVs and MERS-CoV. In 2016, 10 years after the discovery of Rhinolophus BatCoV HKU2 in Chinese horseshoe bats, fatal swine disease outbreaks caused by this virus were reported in southern China. In late 2019, an outbreak of pneumonia emerged in Wuhan, China, and rapidly spread globally, leading to >4,000,000 fatalities so far. Although the genome of SARS-CoV-2 is highly similar to that of SARS-CoV, patient zero and the original source of the pandemic are still unknown. To protect humans from future public health threats, measures should be taken to monitor and reduce the chance of interspecies jumping events, either occurring naturally or through recombineering experiments.

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.

Abstract Although multilocus sequence typing (MLST) is highly discriminatory and useful for outbreak investigations and epidemiological surveillance, it has always been controversial whether clustering and phylogeny inferred from the MLST gene loci can represent the real phylogeny of bacterial strains. In this study, we compare the phylogenetic trees constructed using three approaches, (1) concatenated blocks of homologous sequence shared between the bacterial genomes, (2) genome single-nucleotide polymorphisms (SNP) profile and (3) concatenated nucleotide sequences of gene loci in the corresponding MLST schemes, for 10 bacterial species with >30 complete genome sequences available. Major differences in strain clustering at more than one position were observed between the phylogeny inferred using genome/SNP data and MLST for all 10 bacterial species. Shimodaira-Hasegawa test revealed significant difference between the topologies of the genome and MLST trees for nine of the 10 bacterial species, and significant difference between the topologies of the SNP and MLST trees were present for all 10 bacterial species. Matching Clusters and R-F Clusters metrics showed that the distances between the genome/SNP and MLST trees were larger than those between the SNP and genome trees. Phylogeny inferred from MLST failed to represent genome phylogeny with the same bacterial species.

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.

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.

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.

We analyzed the incidence and the clinical and laboratory characteristics of Staphylococcus lugdunensis urinary tract infections (UTIs) during a 10-year period (2009−2018) and compared them with those of Staphylococcus saprophyticus UTIs. A total of 38 and 162 episodes of S. lugdunensis and S. saprophyticus UTIs were observed. The number of S. saprophyticus UTIs was stable throughout the 10 years, whereas there was an obvious surge in the apparent number of S. lugdunensis UTIs since 2014, coinciding with the commencement of a routine use of MALDI-TOF MS. Univariate analysis showed that male sex (p < 0.001), advanced age (p < 0.001), hospital-acquired infections, (p < 0.001), upper UTI (p < 0.005), polymicrobial infections (p < 0.05), hypertension (p < 0.001), solid-organ malignancies (p < 0.001), renal stones (p < 0.001), urinary stricture (p < 0.05), vesicoureteral reflux (p < 0.001), and presence of a urinary catheter (p < 0.001) were significantly associated with S. lugdunensis UTI. Multivariable analysis revealed that S. lugdunensis UTI was associated with male sex (OR = 6.08, p < 0.05), solid-organ malignancies (OR = 12.27, p < 0.01), and urological system abnormalities (OR = 7.44, p < 0.05). There were significant differences in the patient population affected and predisposing factors between S. lugdunensis and S. saprophyticus UTIs.

ABSTRACT Talaromyces marneffei is the most important thermal dimorphic fungus causing systemic mycosis in Southeast Asia. We report the discovery of a novel partitivirus, Talaromyces marneffei partitivirus-1 (TmPV1). TmPV1 was detected in 7 (12.7%) of 55 clinical T. marneffei isolates. Complete genome sequencing of the seven TmPV1 isolates revealed two double-stranded RNA (dsRNA) segments encoding RNA-dependent RNA polymerase (RdRp) and capsid protein, respectively. Phylogenetic analysis showed that TmPV1 occupied a distinct clade among the members of the genus Gammapartitivirus. Transmission electron microscopy confirmed the presence of isometric, nonenveloped viral particles of 30 to 45 nm in diameter, compatible with partitiviruses, in TmPV1-infected T. marneffei. Quantitative reverse transcription-PCR (qRT-PCR) demonstrated higher viral load of TmPV1 in the yeast phase than in the mycelial phase of T. marneffei. Two virus-free isolates, PM1 and PM41, were successfully infected by purified TmPV1 using protoplast transfection. Mice challenged with TmPV1-infected T. marneffei isolates showed significantly shortened survival time (P < 0.0001) and higher fungal burden in organs than mice challenged with isogenic TmPV1-free isolates. Transcriptomic analysis showed that TmPV1 causes aberrant expression of various genes in T. marneffei, with upregulation of potential virulence factors and suppression of RNA interference (RNAi)-related genes. This is the first report of a mycovirus in a thermally dimorphic fungus. Further studies are required to ascertain the mechanism whereby TmPV1 enhances the virulence of T. marneffei in mice and the potential role of RNAi-related genes in antiviral defense in T. marneffei. IMPORTANCE Talaromyces marneffei (formerly Penicillium marneffei) is the most important thermal dimorphic fungus in Southeast Asia, causing highly fatal systemic penicilliosis in HIV-infected and immunocompromised patients. We discovered a novel mycovirus, TmPV1, in seven clinical isolates of T. marneffei. TmPV1 belongs to the genus Gammapartitivirus of the family Partitiviridae. We showed that TmPV1 enhanced the virulence of T. marneffei in mice, with shortened survival time and higher fungal burden in the organs of mice challenged with TmPV1-infected T. marneffei isolates than in those of mice challenged with virus-free isogenic isolates. Transcriptomics analysis showed that TmPV1 altered the expression of genes involved in various cellular processes in T. marneffei, with upregulation of potential virulence factors and suppression of RNAi machinery which may be involved in antiviral defense. This is the first report of a mycovirus in a thermal dimorphic fungus. The present results offer insights into mycovirus-fungus interactions and pathogenesis of thermal dimorphic fungi.

Penicillium marneffei (synonym: Talaromyces marneffei) is the most important pathogenic thermally dimorphic fungus in China and Southeastern Asia. The HIV/AIDS pandemic, particularly in China and other Southeast Asian countries, has led to the emergence of P. marneffei infection as an important AIDS-defining condition. Recently, we published the genome sequence of P. marneffei. In the P. marneffei genome, 23 polyketide synthase genes and two polyketide synthase-non-ribosomal peptide synthase hybrid genes were identified. This number is much higher than those of Coccidioides immitis and Histoplasma capsulatum, important pathogenic thermally dimorphic fungi in the Western world. Phylogenetically, these polyketide synthase genes were distributed evenly with their counterparts found in Aspergillus species and other fungi, suggesting that polyketide synthases in P. marneffei did not diverge from lineage-specific gene duplication through a recent expansion. Gene knockdown experiments and ultra-high performance liquid chromatography-photodiode array detector/electrospray ionization-quadruple time of flight-mass spectrometry analysis confirmed that at least four of the polyketide synthase genes were involved in the biosynthesis of various pigments in P. marneffei, including melanin, mitorubrinic acid, mitorubrinol, monascorubrin, rubropunctatin, citrinin and ankaflavin, some of which were mycotoxins and virulence factors of the fungus.

Camels carry Middle East respiratory syndrome coronavirus, but little is known about infection age or prevalence. We studied >800 dromedaries of all ages and 15 mother–calf pairs. This syndrome constitutes an acute, epidemic, and time-limited infection in camels

Although PacBio third-generation sequencers have improved the read lengths of genome sequencing which facilitates the assembly of complete genomes, no study has reported success in using PacBio data alone to completely sequence a two-chromosome bacterial genome from a single library in a single run. Previous studies using earlier versions of sequencing chemistries have at most been able to finish bacterial genomes containing only one chromosome with de novo assembly. In this study, we compared the robustness of PacBio RS II, using one SMRT cell and the latest P6-C4 chemistry, with Illumina HiSeq 1500 in sequencing the genome of Burkholderia pseudomallei, a bacterium which contains two large circular chromosomes, very high G+C content of 68–69%, highly repetitive regions and substantial genomic diversity, and represents one of the largest and most complex bacterial genomes sequenced, using a reference genome generated by hybrid assembly using PacBio and Illumina datasets with subsequent manual validation. Results showed that PacBio data with de novo assembly, but not Illumina, was able to completely sequence the B. pseudomallei genome without any gaps or mis-assemblies. The two large contigs of the PacBio assembly aligned unambiguously to the reference genome, sharing >99.9% nucleotide identities. Conversely, Illumina data assembled using three different assemblers resulted in fragmented assemblies (201–366 contigs), sharing only 92.2–100% and 92.0–100% nucleotide identities to chromosomes I and II reference sequences, respectively, with no indication that the B. pseudomallei genome consisted of two chromosomes with four copies of ribosomal operons. Among all assemblies, the PacBio assembly recovered the highest number of core and virulence proteins, and housekeeping genes based on whole-genome multilocus sequence typing (wgMLST). Most notably, assembly solely based on PacBio outperformed even hybrid assembly using both PacBio and Illumina datasets. Hybrid approach generated only 74 contigs, while the PacBio data alone with de novo assembly achieved complete closure of the two-chromosome B. pseudomallei genome without additional costly bench work and further sequencing. PacBio RS II using P6-C4 chemistry is highly robust and cost-effective and should be the platform of choice in sequencing bacterial genomes, particularly for those that are well-known to be difficult-to-sequence.

While dromedaries are the immediate animal source of Middle East Respiratory Syndrome (MERS) epidemic, viruses related to MERS coronavirus (MERS-CoV) have also been found in bats as well as hedgehogs. To elucidate the evolution of MERS-CoV-related viruses and their interspecies transmission pathway, samples were collected from different mammals in China. A novel coronavirus related to MERS-CoV, Erinaceus amurensis hedgehog coronavirus HKU31 (Ea-HedCoV HKU31), was identified from two Amur hedgehogs. Genome analysis supported that Ea-HedCoV HKU31 represents a novel species under Merbecovirus, being most closely related to Erinaceus CoV from European hedgehogs in Germany, with 79.6% genome sequence identity. Compared to other members of Merbecovirus, Ea-HedCoV HKU31 possessed unique non-structural proteins and putative cleavage sites at ORF1ab. Phylogenetic analysis showed that Ea-HedCoV HKU31 and BetaCoV Erinaceus/VMC/DEU/2012 were closely related to NeoCoV and BatCoV PREDICT from African bats in the spike region, suggesting that the latter bat viruses have arisen from recombination between CoVs from hedgehogs and bats. The predicted HKU31 receptor-binding domain (RBD) possessed only one out of 12 critical amino acid residues for binding to human dipeptidyl peptidase 4 (hDPP4), the MERS-CoV receptor. The structural modeling of the HKU31-RBD-hDPP4 binding interphase compared to that of MERS-CoV and Tylonycteris bat CoV HKU4 (Ty-BatCoV HKU4) suggested that HKU31-RBD is unlikely to bind to hDPP4. Our findings support that hedgehogs are an important reservoir of Merbecovirus, with evidence of recombination with viruses from bats. Further investigations in bats, hedgehogs and related animals are warranted to understand the evolution of MERS-CoV-related viruses.

Enzootic nasal tumor virus (ENTV) has two types, ENTV-1 in sheep and ENTV-2 in goats, respectively. In China, the incidence of ENTV-2 related diseases has increased year by year. In this study, we reported an outbreak of ENTV-2 in a commercial goat farm in Qingyuan city, Guangdong province, southern China. A full-length genome of ENTV-2 (designated GDQY2017), with 7479 base pairs, was sequenced. Although GDQY2017 shared the highest nucleotide identity with a Chinese ENTV-2 isolate (ENTV-2CHN4, GenBank accession number KU258873), it possesses distinct genome characteristics undescribed, including a non-continuous 21-nucleotide insertion in the gag gene and a non-continuous 12-nucleotide deletion in the env gene. Notably, most of these indel nucleotide sequences were originated from a Chinese jaagsiekte sheep retrovirus (JSRV) isolate (GenBank accession number DQ838494). In the gag and env genes, GDQY2017 was phylogenetically related to those Chinese ENTV-2 isolates and a Chinese JSRV isolate (DQ838494). For GDQY2017-like viruses, more surveillance work should be made to explain their pathogenicity in goat herds. To our knowledge, this study represents the first to demonstrate the circulating pattern of ENTV-2 in Guangdong province, China, which will help to better understand the epidemiology and genetic diversity of ENTV-2.

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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.

Bats are a unique group of mammals of the order Chiroptera. They are highly diversified and are the group of mammals with the second largest number of species. Such highly diversified cell types and receptors facilitate them to be potential hosts of a large variety of viruses. Bats are the only group of mammals capable of sustained flight, which enables them to disseminate the viruses they harbor and enhance the chance of interspecies transmission. This article aims at reviewing the various aspects of the global epidemiology of bat coronaviruses (CoVs). Before the SARS epidemic, bats were not known to be hosts for CoVs. In the last 15 years, bats have been found to be hosts of >30 CoVs with complete genomes sequenced, and many more if those without genome sequences are included. Among the four CoV genera, only alphaCoVs and betaCoVs have been found in bats. As a whole, both alphaCoVs and betaCoVs have been detected from bats in Asia, Europe, Africa, North and South America and Australasia; but alphaCoVs seem to be more widespread than betaCoVs, and their detection rate is also higher. For betaCoVs, only those from subgenera Sarbecovirus, Merbecovirus, Nobecovirus and Hibecovirus have been detected in bats. Most notably, horseshoe bats are the reservoir of SARS-CoV, and several betaCoVs from subgenus Merbecovirus are closely related to MERS-CoV. In addition to the interactions among various bat species themselves, bat⁻animal and bat⁻human interactions, such as the presence of live bats in wildlife wet markets and restaurants in Southern China, are important for interspecies transmission of CoVs and may lead to devastating global outbreaks.

Human adenovirus type 4 (HAdV-E4), which is intriguingly limited to military populations, causes acute respiratory disease with demonstrated morbidity and mortality implications. This respiratory pathogen contains genome identity with chimpanzee adenoviruses, indicating zoonotic origins. A signature of these “old„ HAdV-E4 is the absence of a critical replication motif, NF-I, which is found in all HAdV respiratory pathogens and most HAdVs. However, our recent survey of flu-like disease in children in Hong Kong reveals that the emergent HAdV-E4 pathogens circulating in civilian populations contain NF-I, indicating recombination and reflecting host-adaptation that enables the “new„ HAdV-E4 to replicate more efficiently in human cells and foretells more potential HAdV-E4 outbreaks in immune-naïve civilian populations. Special attention should be paid by clinicians to this emergent and recombinant HAdV-E4 circulating in civilian populations.

Owing to the highly similar phenotypic profiles, protein spectra and 16S rRNA gene sequences observed between three pairs of Tsukamurella species (Tsukamurella pulmonis/Tsukamurella spongiae, Tsukamurella tyrosinosolvens/Tsukamurella carboxydivorans and Tsukamurella pseudospumae/Tsukamurella sunchonensis), we hypothesize that and the six Tsukamurella species may have been misclassified and that there may only be three Tsukamurella species. In this study, we characterized the type strains of these six Tsukamurella species by tradition DNA-DNA hybridization (DDH) and digital DDH after genome sequencing to determine their exact taxonomic positions. Traditional DDH showed 81.2±0.6% to 99.7±1.0% DNA-DNA relatedness between the two Tsukamurella species in each of the three pairs, which was above the threshold for same species designation. Digital DDH based on Genome-To-Genome Distance Calculator (GGDC) and Average Nucleotide Identity (ANI) for the three pairs also showed similarity results in the range of 82.3-92.9% and 98.1-99.1% respectively, in line with results of traditional DDH. Based on these evidence and according to Rules 23a and 42 of the Bacteriological Code, we propose that T. spongiae Olson et al. 2007 should be reclassified as a later heterotypic synonym of T. pulmonis Yassin et al. 1996, T. carboxydivorans Park et al. 2009 as a later heterotypic synonym of T. tyrosinosolvens Yassin et al. 1997, and T. sunchonensis Seong et al. 2008 as a later heterotypic synonym of T. pseudospumae Nam et al. 2004. With the advancement of genome sequencing technologies, classification of bacterial species can be readily achieved by digital DDH than traditional DDH.

The drastic increase in the number of coronaviruses discovered and coronavirus genomes being sequenced have given us an unprecedented opportunity to perform genomics and bioinformatics analysis on this family of viruses. Coronaviruses possess the largest genomes (26.4 to 31.7 kb) among all known RNA viruses, with G + C contents varying from 32% to 43%. Variable numbers of small ORFs are present between the various conserved genes (ORF1ab, spike, envelope, membrane and nucleocapsid) and downstream to nucleocapsid gene in different coronavirus lineages. Phylogenetically, three genera, Alphacoronavirus, Betacoronavirus and Gammacoronavirus, with Betacoronavirus consisting of subgroups A, B, C and D, exist. A fourth genus, Deltacoronavirus, which includes bulbul coronavirus HKU11, thrush coronavirus HKU12 and munia coronavirus HKU13, is emerging. Molecular clock analysis using various gene loci revealed that the time of most recent common ancestor of human/civet SARS related coronavirus to be 1999-2002, with estimated substitution rate of 4´10-4 to 2´10-2 substitutions per site per year. Recombination in coronaviruses was most notable between different strains of murine hepatitis virus (MHV), between different strains of infectious bronchitis virus, between MHV and bovine coronavirus, between feline coronavirus (FCoV) type I and canine coronavirus generating FCoV type II, and between the three genotypes of human coronavirus HKU1 (HCoV-HKU1). Codon usage bias in coronaviruses were observed, with HCoV-HKU1 showing the most extreme bias, and cytosine deamination and selection of CpG suppressed clones are the two major independent biological forces that shape such codon usage bias in coronaviruses.

After human coronaviruses OC43, 229E and NL63, human coronavirus HKU1 (HCoV-HKU1) is the fourth human coronavirus discovered. HCoV-HKU1 is a group 2a coronavirus that is still not cultivable. The G + C contents of HCoV-HKU1 genomes are 32%, the lowest among all known coronaviruses with complete genome sequences available. Among all coronaviruses, HCoV-HKU1 shows the most extreme codon usage bias, attributed most importantly to severe cytosine deamination. All HCoV-HKU1 genomes contain unique tandem copies of a 30-base acidic tandem repeat of unknown function at the N-terminus of nsp3 inside the acidic domain upstream of papain-like protease 1. Three genotypes, A, B and C, of HCoV-HKU1 and homologous recombination among their genomes, are observed. The incidence of HCoV-HKU1 infections is the highest in winter. Similar to other human coronaviruses, HCoV-HKU1 infections have been reported globally, with a median (range) incidence of 0.9 (0 – 4.4) %. HCoV-HKU1 is associated with both upper and lower respiratory tract infections that are mostly self-limiting. The most common method for diagnosing HCoV-HKU1 infection is RT-PCR or real-time RT-PCR using RNA extracted from respiratory tract samples such as nasopharyngeal aspirates (NPA). Both the pol and nucleocapsid genes have been used as the targets for amplification. Monoclonal antibodies have been generated for direct antigen detection in NPA. For antibody detection, Escherichia coli BL21 and baculovirus-expressed recombinant nucleocapsid of HCoV-HKU1 have been used for IgG and IgM detection in sera of patients and normal individuals, using Western blot and enzyme-linked immunoassay.

Talaromyces marneffei is a thermally dimorphic fungus causing systemic infections in patients positive for HIV or other immunocompromised statuses. Analysis of its ~28.9 Mb draft genome and additional transcriptomic, proteomic and metabolomic studies revealed mechanisms for environmental adaptations and virulence. Meiotic genes and genes for pheromone receptors, enzymes which process pheromones, and proteins involved in pheromone response pathway are present, indicating its possibility as a heterothallic fungus. Among the 14 Mp1p homologs, only Mp1p is a virulence factor binding a variety of host proteins, fatty acids and lipids. There are 23 polyketide synthase genes, one for melanin and two for mitorubrinic acid/mitorubrinol biosynthesis, which are virulence factors. Another polyketide synthase is for biogenesis of the diffusible red pigment, which consists of amino acid conjugates of monascorubin and rubropunctatin. Novel microRNA-like RNAs (milRNAs) and processing proteins are present. The dicer protein, dcl-2, is required for biogenesis of two milRNAs, PM-milR-M1 and PM-milR-M2, which are more highly expressed in hyphal cells. Comparative transcriptomics showed that tandem repeat-containing genes were overexpressed in yeast phase, generating protein polymorphism among cells, evading host’s immunity. Comparative proteomics between yeast and hyphal cells revealed that glyceraldehyde-3-phosphate dehydrogenase, up-regulated in hyphal cells, is an adhesion factor for conidial attachment.