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We showed that severe acute respiratory syndrome coronavirus 2 is probably a novel recombinant virus. Its genome is closest to that of severe acute respiratory syndrome-related coronaviruses from horseshoe bats, and its receptor-binding domain is closest to that of pangolin viruses. Its origin and direct ancestral viruses have not been identified.

Severe acute respiratory syndrome coronavirus 2 did not replicate efficiently in 13 bat cell lines, whereas severe acute respiratory syndrome coronavirus replicated efficiently in kidney cells of its ancestral host, the Rhinolophus sinicus bat, suggesting different evolutionary origins. Structural modeling showed that RBD/RsACE2 binding may contribute to the differential cellular tropism.

The mortality of human infection by influenza A/H5N1 virus can exceed 80%. The high mortality and its poor response to the neuraminidase inhibitor oseltamivir have been attributed to uncontrolled virus-induced cytokine storm. We challenged BALB/c mice with 1,000 LD₅₀ of influenza A/Vietnam/1194/04. Survival, body weight, histopathology, inflammatory markers, viral loads, T lymphocyte counts, and neutralizing antibody response were documented in infected mice treated individually or in combination with zanamvir, celecoxib, gemfibrozil, and mesalazine. To imitate the real-life scenario, treatment was initiated at 48 h after viral challenge. There were significant improvements in survival rate (P = 0.02), survival time (P < 0.02), and inflammatory markers (P < 0.01) in the group treated with a triple combination of zanamivir, celecoxib, and mesalazine when compared with zanamivir alone. Zanamivir with or without immunomodulators reduced viral load to a similar extent. Insignificant prolongation of survival was observed when individual agents were used alone. Significantly higher levels of CD4⁺ and CD8⁺ T lymphocytes and less pulmonary inflammation were also found in the group receiving triple therapy. Zanamivir alone reduced viral load but not inflammation and mortality. The survival benefits of adding celecoxib and mesalazine to zanamivir could be caused by their synergistic effects in reducing cytokine dysfunction and preventing apoptosis. Combinations of a neuraminidase inhibitor with these immunomodulators should be considered in randomized controlled treatment trials of patients suffering from H5N1 infection.

Penicillium marneffei is the most important thermal dimorphic fungus causing systemic mycosis in China and Southeast Asia. While miRNAs are increasingly recognized for their roles in post-transcriptional regulation of gene expression in animals and plants, miRNAs in fungi were less well studied and their potential roles in fungal dimorphism were largely unknown. Based on P. marneffei genome sequence, we hypothesize that miRNA-like RNAs (milRNAs) may be expressed in the dimorphic fungus. We attempted to identify milRNAs in P. marneffei in both mycelial and yeast phase using high-throughput sequencing technology. Small RNAs were more abundantly expressed in mycelial than yeast phase. Sequence analysis revealed 24 potential milRNA candidates, including 17 candidates in mycelial and seven in yeast phase. Two genes, dcl-1 and dcl-2, encoding putative Dicer-like proteins and the gene, qde-2, encoding Argonaute-like protein, were identified in P. marneffei. Phylogenetic analysis showed that dcl-2 of P. marneffei was more closely related to the homologues in other thermal dimorphic pathogenic fungi than to Penicillium chrysogenum and Aspergillus spp., suggesting the co-evolution of dcl-2 among the thermal dimorphic fungi. Moreover, dcl-2 demonstrated higher mRNA expression levels in mycelial than yeast phase by 7 folds (P<0.001). Northern blot analysis confirmed the expression of two milRNAs, PM-milR-M1 and PM-milR-M2, only in mycelial phase. Using dcl-1(KO), dcl-2(KO), dcl(DKO) and qde-2(KO) deletion mutants, we showed that the biogenesis of both milRNAs were dependent on dcl-2 but not dcl-1 or qde-2. The mRNA expression levels of three predicted targets of PM-milR-M1 were upregulated in knockdown strain PM-milR-M1 (KD), supporting regulatory function of milRNAs. Our findings provided the first evidence for differential expression of milRNAs in different growth phases of thermal dimorphic fungi and shed light on the evolution of fungal proteins involved in milRNA biogenesis and possible role of post-transcriptional control in governing thermal dimorphism.

Systemic dimorphic fungi cause more than one million new infections each year, ranking them among the significant public health challenges currently encountered. Penicillium marneffei is a systemic dimorphic fungus endemic to Southeast Asia. The temperature-dependent dimorphic phase transition between mycelium and yeast is considered crucial for the pathogenicity and transmission of P. marneffei, but the underlying mechanisms are still poorly understood. Here, we re-sequenced P. marneffei strain PM1 using multiple sequencing platforms and assembled the genome using hybrid genome assembly. We determined gene expression levels using RNA sequencing at the mycelial and yeast phases of P. marneffei, as well as during phase transition. We classified 2,718 genes with variable expression across conditions into 14 distinct groups, each marked by a signature expression pattern implicated at a certain stage in the dimorphic life cycle. Genes with the same expression patterns tend to be clustered together on the genome, suggesting orchestrated regulations of the transcriptional activities of neighboring genes. Using qRT-PCR, we validated expression levels of all genes in one of clusters highly expressed during the yeast-to-mycelium transition. These included madsA, a gene encoding MADS-box transcription factor whose gene family is exclusively expanded in P. marneffei. Over-expression of madsA drove P. marneffei to undergo mycelial growth at 37°C, a condition that restricts the wild-type in the yeast phase. Furthermore, analyses of signature expression patterns suggested diverse roles of secreted proteins at different developmental stages and the potential importance of non-coding RNAs in mycelium-to-yeast transition. We also showed that RNA structural transition in response to temperature changes may be related to the control of thermal dimorphism. Together, our findings have revealed multiple molecular mechanisms that may underlie the dimorphic transition in P. marneffei, providing a powerful foundation for identifying molecular targets for mechanism-based interventions.

The genome of P. marneffei, the most important thermal dimorphic fungus causing respiratory, skin and systemic mycosis in China and Southeast Asia, possesses 23 polyketide synthase (PKS) genes and 2 polyketide synthase nonribosomal peptide synthase hybrid (PKS-NRPS) genes, which is of high diversity compared to other thermal dimorphic pathogenic fungi. We hypothesized that the yellow pigment in the mold form of P. marneffei could also be synthesized by one or more PKS genes. All 23 PKS and 2 PKS-NRPS genes of P. marneffei were systematically knocked down. A loss of the yellow pigment was observed in the mold form of the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants. Sequence analysis showed that PKS11 and PKS12 are fungal non-reducing PKSs. Ultra high performance liquid chromatography-photodiode array detector/electrospray ionization-quadruple time of flight-mass spectrometry (MS) and MS/MS analysis of the culture filtrates of wild type P. marneffei and the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants showed that the yellow pigment is composed of mitorubrinic acid and mitorubrinol. The survival of mice challenged with the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants was significantly better than those challenged with wild type P. marneffei (P<0.05). There was also statistically significant decrease in survival of pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants compared to wild type P. marneffei in both J774 and THP1 macrophages (P<0.05). The yellow pigment of the mold form of P. marneffei is composed of mitorubrinol and mitorubrinic acid. This represents the first discovery of PKS genes responsible for mitorubrinol and mitorubrinic acid biosynthesis. pks12 and pks11 are probably responsible for sequential use in the biosynthesis of mitorubrinol and mitorubrinic acid. Mitorubrinol and mitorubrinic acid are virulence factors of P. marneffei by improving its intracellular survival in macrophages.

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.

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.

Hepatitis E virus (HEV) is a major cause of viral hepatitis globally. Zoonotic HEV is an important cause of chronic hepatitis in immunocompromised patients. The rapid identification of novel HEV variants and accumulating sequence information has prompted significant changes in taxonomy of the family Hepeviridae. This family includes two genera: Orthohepevirus, which infects terrestrial vertebrates, and Piscihepevirus, which infects fish. Within Orthohepevirus, there are four species, A–D, with widely differing host range. Orthohepevirus A contains the HEV variants infecting humans and its significance continues to expand with new clinical information. We now recognize eight genotypes within Orthohepevirus A: HEV1 and HEV2, restricted to humans; HEV3, which circulates among humans, swine, rabbits, deer and mongooses; HEV4, which circulates between humans and swine; HEV5 and HEV6, which are found in wild boars; and HEV7 and HEV8, which were recently identified in dromedary and Bactrian camels, respectively. HEV7 is an example of a novel genotype that was found to have significance to human health shortly after discovery. In this review, we summarize recent developments in HEV molecular taxonomy, epidemiology and evolution and describe the discovery of novel camel HEV genotypes as an illustrative example of the changes in this field.

Talaromyces (Penicillium) marneffei is an important pathogenic thermally dimorphic fungus causing systemic mycosis in Southeast Asia. The clinical significance of T. marneffei became evident when the human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome epidemic arrived in Southeast Asia in 1988. Subsequently, a decline in the incidence of T. marneffei infection among HIV-infected patients was seen in regions with access to highly active antiretroviral therapy and other control measures for HIV. Since the 1990s, an increasing number of T. marneffei infections have been reported among non-HIV-infected patients with impaired cell-mediated immunity. Their comorbidities included primary adult-onset immunodeficiency due to anti-interferon-gamma autoantibodies and secondary immunosuppressive conditions including other autoimmune diseases, solid organ and hematopoietic stem cell transplantations, T-lymphocyte-depleting immunsuppressive drugs and novel anti-cancer targeted therapies such as anti-CD20 monoclonal antibodies and kinase inhibitors. Moreover, improved immunological diagnostics identified more primary immunodeficiency syndromes associated with T. marneffei infection in children. The higher case-fatality rate of T. marneffei infection in non-HIV-infected than HIV-infected patients might be related to delayed diagnosis due to the lack of clinical suspicion. Correction of the underlying immune defects and early use of antifungals are important treatment strategies. Clinicians should be familiar with the changing epidemiology and clinical management of T. marneffei infection among non-HIV-infected patients.