A Broad-specificity Neutralizing Nanobody against Hepatitis E Virus Capsid Protein.

Hepatitis E virus (HEV) is a worldwide zoonotic and public health concern. The study of HEV biology is helpful for designing viral vaccines and drugs. Nanobodies have recently been considered appealing materials for viral biological research. In this study, a Bactrian camel was immunized with capsid proteins from different genotypes (1, 3, 4, and avian) of HEV. Then, a phage library (6.3 × 108 individual clones) was constructed using peripheral blood lymphocytes from the immunized camel, and 12 nanobodies against the truncated capsid protein of genotype 3 HEV (g3-p239) were screened. g3-p239-Nb55 can cross-react with different genotypes of HEV and block Kernow-C1/P6 HEV from infecting HepG2/C3A cells. To our knowledge, the epitope recognized by g3-p239-Nb55 was determined to be a novel conformational epitope located on the surface of viral particles and highly conserved among different mammalian HEV isolates. Next, to increase the affinity and half-life of the nanobody, it was displayed on the surface of ferritin, which can self-assemble into a 24-subunit nanocage, namely, fenobody-55. The affinities of fenobody-55 to g3-p239 were ∼20 times greater than those of g3-p239-Nb55. In addition, the half-life of fenobody-55 was nine times greater than that of g3-p239-Nb55. G3-p239-Nb55 and fenobody-55 can block p239 attachment and Kernow-C1/P6 infection of HepG2/C3A cells. Fenobody-55 can completely neutralize HEV infection in rabbits when it is preincubated with nonenveloped HEV particles. Our study reported a case in which a nanobody neutralized HEV infection by preincubation, identified a (to our knowledge) novel and conserved conformational epitope of HEV, and provided new material for researching HEV biology.

Hepatitis E virus causes apoptosis of ovarian cells in hens and resulting in a decrease in egg production.

Previous studies have shown that avian hepatitis E virus (HEV) decreases egg production by 10-40% in laying hens, but have not fully elucidated the mechanism of there. In this study, we evaluated the replication of avian HEV in the ovaries of laying hens and the mechanism underlying the decrease in egg production. Forty 150-days-old commercial laying hens were randomly divided into 2 groups of 20 hens each. A total of 1 mL (104GE) of avian HEV stock was inoculated intravenously into each chicken in the experimental group, with 20 chickens in the other group serving as negative controls. Five chickens from each group were necropsied weekly for histopathological examination. The pathogenicity of avian HEV has been characterized by seroconversion, viremia, fecal virus shedding, ovarian lesions, and decreased egg production. Both positive and negative-strand avian HEV RNA, and ORF2 antigens can be detected in the ovaries, suggesting that avian HEV can replicate in the ovaries and serve as an important extrahepatic replication site. The ovaries of laying hens underwent apoptosis after avian HEV infection. These results indicate that avian HEV infection and replication in ovarian tissues cause structural damage to the cells, leading to decreased egg production.

Dissecting the genome sequence of a clinical isolated Cunninghamella bertholletiae Z2 strain with rich cytochrome P450 enzymes (Article).

Mucormycosis is receiving much more attention because of its high morbidity and extremely high mortality rate in immunosuppressed populations. In this study, we isolated a Cunnignhamella bertholletiae Z2 strain from a skin lesion of a 14 year, 9 months old girl with acute lymphoblastic leukemia who die of infection from the Z2 strain. Genome sequencing was performed after isolation and amplification of the Z2 strain to reveal potential virulence factors and pathogenic mechanisms. The results showed that the genome size of the Z2 strain is 30.9 Mb with 9213 genes. Mucoral specific virulence factor genes found are ARF, CalN, and CoTH, while no gliotoxin biosynthesis gene cluster was found, which is a known virulence factor in Aspergillus fumigatus adapted to the environment. The Z2 strain was found to have 69 cytochrome P450 enzymes, which are potential drug resistant targets. Sensitivity testing of Z2 showed it was only inhibited by amphotericin B and posaconazole. Detailed genomic information of the C. bertholletiae Z2 strain may provide useful data for treatment.

Amino acids 1811-1960 of myosin heavy chain 9 is involved in murine gammaherpesvirus 68 infection.

Myosin heavy chain 9 (MYH9) has been identified as a crucial factor in gammaherpesvirus infection. Murine gammaherpesvirus 68 (MHV-68) was used as an appropriate viral model for investigating gammaherpesviruses in vivo and developing antiviral treatments. However, the roles of MYH9 in MHV-68 infection have not been documented. In the study, the relationship between the expression of MYH9 and MHV-68 infection and MYH9 as the antiviral target were analyzed. The results revealed that MYH9 was enriched on the cell surface and co-localized with MHV-68 upon viral infection. Knocking down MYH9 with siRNA or using the specific inhibitor of MYH9 activity, Blebbistatin, resulted in the decreasing of MHV-68 infection. Furthermore, polyclonal antibodies against MYH9 reduced infection by approximately 74% at a dose of 100 µg/ml. The study determined that MYH9 contributes to MHV-68 infection by interacting with viral glycoprotein 150 (gp150) in the BHK-21 cell membrane. The specific region of MYH9, amino acids 1811-1960 (C-150), was identified as the key domain involved in the interaction with MHV-68 gp150 and was found to inhibit MHV-68 infection. Moreover, C-150 was also shown to decrease HSV-1 infection in Vero cells by approximately 73%. Both C-150 and Blebbistatin were found to inhibit MHV-68 replication and reduce histopathological lesions in vivo in C57BL/6J mice. Taken together, these findings suggested that MYH9 is crucial for MHV-68 infection through its interaction with viral gp150 and that C-150 may be a promising antiviral target for inhibiting MHV-68 infection in vitro and in vivo.

Development of a novel competitive ELISA based on nanobody-horseradish peroxidase fusion protein for rapid detection of antibodies against avian hepatitis E virus.

Avian hepatitis E virus (avian HEV) increases poultry mortality and decreases egg production, leading to huge economic losses worldwide. However, there is no effective serological test for avian HEV. Researchers previously created a testing platform using the nanobody (Nb)-horseradish peroxidase (HRP) fusion protein as an ultrasensitive probe to develop competitive ELISA (cELISA) to detect antibodies against different animal viruses. In this study, a rapid and reliable cELISA was developed to test for antibodies against avian HEV using the same platform. Six anti-avian HEV capsid protein nanobodies were selected from an immunized Bactrian camel using phage display technology. The avian HEV-Nb49-HRP fusion protein was expressed and used as a probe for developing a cELISA assay to test for avian HEV antibodies. The cut-off value of the developed cELISA was 22.0%. There was no cross-reaction with other anti-avian virus antibodies, suggesting that the cELISA had good specificity. The coefficients of variation were 0.91% to 4.21% (intra-assay) and 1.52% to 6.35% (inter-assay). Both cELISA and indirect ELISA showed a consistency of 86.7% (kappa = 0.738) for clinical chicken serum samples, and coincidence between cELISA and Western blot was 96.0% (kappa = 0.919). The epitope recognized by Nb49 was located in aa 593-604 of the avian HEV capsid protein, and the peptide (TFPS) in aa 601-604 was essential for binding. The novel cELISA is a saving cost, rapid, useful, and reliable assay for the serological investigation of avian HEV. More importantly, the peptide TFPS may be crucial to immunodominant antigen composition and protection.

Paenibacillus hamazuiensis sp. nov., a bacterium isolated from Hamazui hot spring in Yunnan province, south-west China.

A bacterial strain, Gram-positive, aerobic, rod-shaped, motile, designated YIM B00624T which was isolated from a Hamazui hot spring in Tengchong, Yunnan province, south-west China. The strain grew well on International Streptomyces Project (ISP) 2 medium and colonies were creamy yellow, flat and circular. The results of 16S rRNA gene sequence similarity analysis showed that strain YIM B00624T was closely related to the type strain of Paenibacillus filicis S4T (95.9%). The main menaquinone of strain YIM B00624T was menaquinone-7 (MK-7) and major fatty acids were anteiso-C15:0, anteiso-C17:0 and C16:0. The isolate contained meso-diaminopimelic acid as the diagnostic diamino acid and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine and four unidentified glycolipids. The DNA G+C content of strain YIM B00624T was 53.4 mol%. Based on physiological, phenotypic and chemotaxonomic data, strain YIM B00624T belongs to a novel species of the genus Paenibacillus, for which the name Paenibacillus hamazuiensis sp. nov. is proposed. The type strain is YIM B00624T (= CGMCC 1.19245T = KCTC 43365T).

Thermus brevis sp. nov., a moderately thermophilic bacterium isolated from a hot spring microbial mat.

Three closely related, facultative anaerobic, Gram-stain-negative, twitching motile, short rod-shaped, non-endospore-forming, moderately thermophilic bacteria, designated strains SYSU G05001T, SYSU G05003 and SYSU G05004, were isolated from a hot spring microbial mat, collected from Rehai National Park, Tengchong, Yunnan Province, south-western China. The results of phylogenetic analysis based on the 16S rRNA gene sequences indicated that these three strains were closely related to Thermus scotoductus SE-1T (97.97, 98.18, 97.90 % sequence similarity). Whole genome sequencing and polyphasic taxonomic approach were used to determine the genomic profile and taxonomic status of the novel strain SYSU G05001T. Cell growth occurred at 37-80 °C (optimum, 55 °C), pH 6.0-8.0 (optimum, pH 7.0) and with 0-3.0 % (w/v) NaCl (optimum, 1%). Thiosulfate enhanced cell growth. MK-8 was the predominant menaquinone. The major cellular fatty acids included iso-C15 : 0, iso-C17 : 0 and anteiso-C15 : 0. The major polar lipids were consisted of aminophospholipid, glycolipid and phospholipids. The whole genome of strain SYSU G05001T consisted of 2.55 Mbp and the DNA G+C content was 64.94 mol%. The average nucleotide identity (≤94.95 %) and digital DNA-DNA hybridization (≤62.3 %) values between strain SYSU G05001T and other members of the genus Thermus were all lower than the threshold values recommended for distinguishing novel prokaryotic species. On the basis of the presented polyphasic evidence and genotypic data, it is proposed that strain SYSU G05001T (=KCTC 82627T=MCCC 1K06118T) represents a novel species of the genus Thermus, for which the name Thermus brevis sp. nov. is proposed.

Caldalkalibacillus salinus sp. nov., isolated from a salt lake in Xinjiang, northwest China.

A novel Gram-stain-negative, aerobic, motile and rod-shaped bacterium, designated as strain YIM B00319T, was isolated from a sediment sample obtained from Wuzunbulake salt Lake in Xinjiang Uygur Autonomous Region, northwest China. Phylogenetic analysis based on 16S rRNA gene sequences along with the whole genome showed that strain YIM B00319T belongs to the family Bacillaceae and was most closely related to Bacillus horti K13T and Caldalkalibacillus mannanilyticus JCM 10596T, with sequence similarities of 95.7% and 94.6%, respectively. The genome of strain YIM B00319T was 3.77 Mbp with a DNA G + C content of 43%. Strain YIM B00319T grew at 15-45 ℃, pH 7.0-9.5 and with 3-11% (w/v) NaCl. The major respiratory quinone of strain YIM B00319T was MK-7, and the major fatty acids (> 10%) were iso-C15:0, anteiso-C15:0, and summed feature 3 (C16:1 ω6c and/or C16:1 ω7c). The main polar lipids were phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and diphosphatidylglycerol (DPG). The cell-wall peptidoglycan contained meso-diaminopimelic acid. On the basis of the phenotypic, chemotaxonomic, genomic, and phylogenetic information, strain YIM B00319T represents a novel species of the genus Caldalkalibacillus, for which the name Caldalkalibacillus salinus sp. nov. is proposed. The type strain is YIM B00319T (= CGMCC 1.18750T = NBRC 115338T).

Janibacter endophyticus sp. nov., an Endophytic Actinobacterium Isolated from the Root of Paris polyphylla Smith var. Yunnanensis.

A novel endophytic actinobacterium, designated as strain YIM B02568T, was isolated from the root of Paris polyphylla Smith var. Yunnanensis obtained from Yunnan Province, southwest China. Strain YIM B02568T was characterized using a polyphasic approach. Phylogenetic analysis indicated that this isolate belonged to the genus Janibacter. The 16S rRNA gene sequence similarity values of strain YIM B02568T to the type strains of members of this genus ranged from 95.8 to 98.6%. However, overall genome relatedness indices were significantly lower than the widely accepted species-defined threshold. The cell wall of strain YIM B02568T contained meso-diaminopimelic acid. The major menaquinone was MK-8(H4). The main polar lipids were phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylinositol. The major cellular fatty acids were comprised of iso-C16:0 and C18:1 ω9c. The DNA G + C content was 71.6 mol%. Based on the data from the polyphasic studies, we propose that strain YIM B02568T represents a novel species within the genus Janibacter, Janibacter endophyticus sp. nov. The type strain is YIM B02568T (= JCM 34639T = CGMCC 1.18658T).

Glycomyces salinus sp. nov., an actinomycete isolated from a hypersaline habitat.

A Gram-stain-positive, aerobic, nonmotile actinobacterium, designated strain YIM 93776T, was isolated from a saline sediment sample collected from Aiding Lake in Xinjiang Uygur Autonomous Region, Northwest China. Phylogenetic analysis based on the 16S rRNA gene sequences showed that strain YIM 93776T was affiliated to the genus Glycomyces and was closely related to Glycomyces albus TRM 49136T (97.6% sequence similarity), Glycomyces lacisalsi XHU 5089T (97.0%) and Glycomyces anabasis EGI 6500139T (96.2%). The cell wall contained meso-diaminopimelic acid and the whole-cell hydrolysates sugars were galactose, mannose, arabinase, glucose and ribose. The predominant menaquinones were MK-9 (H4) and MK-10 (H4). Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, two phosphatidylglyceride, two unidentified phospholipids and two unidentified polar lipids were detected in the polar lipid extracts. Major fatty acids were anteiso-C17:0, iso-C15:0, iso-C16:0, anteiso-C15:0 and anteiso C17:1 A. The draft genome sequence of strain YIM 93776T was 5.37 Mbp in size with 69.5% DNA G + C content. The dDDH and ANI values between strain YIM 93776T and related neighbours were 25.0-34.3% and 77.3-79.8%, respectively. On the basis of morphological, chemotaxonomic and phylogenetic evidence, strain YIM 93776T; therefore, represents a novel species, for which the name Glycomyces salinus sp. nov. is proposed. The type strain is YIM 93776T (= KCTC 49430T = CGMCC 4.7685T).

Porcine Reproductive and Respiratory Syndrome Virus Promotes SLA-DR-Mediated Antigen Presentation of Nonstructural Proteins To Evoke a Nonneutralizing Antibody Response In Vivo.

The humoral immune response against porcine reproductive and respiratory syndrome virus (PRRSV) infection is characterized by a rapid induction of nonneutralizing antibodies (non-NAbs) against nonstructural proteins (NSPs). Here, we systematically investigated the potential mechanism for the induction of PRRSV NSP-specific non-NAbs. Our data suggested that PRRSV NSP-specific antibodies appeared within 10 days after PRRSV infection in vivo In the in vitro model, functional upregulation of swine leukocyte antigen (SLA)-DR was observed in bone marrow-derived dendritic cells (BMDCs) and porcine alveolar macrophages (PAMs), whereas remarkable inhibition at the mRNA level was observed after infection by both PRRSV-1 and PRRSV-2 isolates. Notably, the inconsistency in SLA-DR expression between the mRNA and protein levels resulted from deubiquitination of SLA-DR via the ovarian tumor (OTU) domain of PRRSV NSP2, which inhibited ubiquitin-mediated degradation. Moreover, mass spectrometry-based immunopeptidome analysis identified immunopeptides originating from multiple PRRSV NSPs within SLA-DR of PRRSV-infected BMDCs. Meanwhile, these PRRSV NSP-derived immunopeptides could be specifically recognized by serum from PRRSV-infected piglets. Notably, certain NSP-derived immunopeptides characterized in vitro could be identified from PAMs or hilar lymph nodes from PRRSV-infected piglets. More importantly, an in vitro neutralizing assay indicated that serum antibodies against NSP immunopeptides were unable to neutralize PRRSV in vitro Conversely, certain structural protein (SP)-derived immunopeptides were identified and could be recognize by pig hyperimmune serum against PRRSV, which further indicates that the NSP-derived antibody response is nonprotective in vivo In conclusion, our data suggested that PRRSV infection interferes with major histocompatibility complex class II (MHC-II) molecule-mediated antigen presentation in antigen-presenting cells (APCs) via promoting SLA-DR expression to present immunopeptides from PRRSV NSPs, which contributes to the induction of non-NAbs in vivoIMPORTANCE PRRSV has haunted the swine industry for over 30 years since its emergence. Besides the limited efficacy of PRRSV modified live vaccines (MLVs) against heterogeneous PRRSV isolates, rapid induction of nonneutralizing antibodies (non-NAbs) against PRRSV NSPs after MLV immunization or wild-strain infection is one of the reasons why development of an effective vaccine has been hampered. By using in vitro-generated BMDCs as models to understand the antigen presentation process of PRRSV, we obtained data indicating that PRRSV infection of BMDCs promotes functional SLA-DR upregulation to present PRRSV NSP-derived immunopeptides for evoking a non-NAb response in vivo Our work not only uncovered a novel mechanism for interference in host antigen presentation by PRRSV but also revealed a novel insight for understanding the rapid production of nonneutralizing antibodies against PRRSV NSPs, which may have benefit for developing an effective vaccine against PRRSV in the future.

Synthetic Peptides Containing Three Neutralizing Epitopes of Genotype 4 Swine Hepatitis E Virus ORF2 induced Protection against Swine HEV Infection in Rabbit.

Genotype 4 hepatitis E virus (HEV) is a zoonotic pathogen transmitted to humans through food and water. Previously, three genotype 4 swine HEV ORF2 peptides (407EPTV410, 410VKLYTS415, and 458PSRPF462) were identified as epitopes of virus-neutralizing monoclonal antibodies that partially blocked rabbit infection with swine HEV. Here, individual and tandem fused peptides were synthesized, conjugated to keyhole limpet hemocyanin (KLH), then evaluated for immunoprotection of rabbits against swine HEV infection. Forty New Zealand White rabbits were randomly assigned to eight groups; groups 1 thru 5 received three immunizations with EPTV-KLH, VKLYTS-KLH, PSRPF-KLH, EPTVKLYTS-KLH, or EPTVKLYTSPSRPF-KLH, respectively; group 6 received truncated swine HEV ORF2 protein (sp239), and group 7 received phosphate-buffered saline. After an intravenous swine HEV challenge, all group 7 rabbits exhibited viremia and fecal virus shedding by 2-4 weeks post challenge (wpc), seroconversion by 4-9 wpc, elevated alanine aminotransferase (ALT) at 2 wpc, and severe liver lymphocytic venous periphlebitis. Only 1-2 rabbits/group in groups 1-4 exhibited delayed viremia, fecal shedding, seroconversion, increased ALT levels, and slight liver lymphocytic venous periphlebitis; groups 5-6 showed no pathogenic effects. Collectively, these results demonstrate that immunization with a polypeptide containing three genotype 4 HEV ORF2 neutralizing epitopes completely protected rabbits against swine HEV infection.

Co-infection with avian hepatitis E virus and avian leukosis virus subgroup J as the cause of an outbreak of hepatitis and liver hemorrhagic syndromes in a brown layer chicken flock in China.

Hens of a commercial Hy-line brown layer flock in China exhibited increased mortality and decreased egg production at 47 wk of age. From 47 to 57 wk, average weekly mortality increased from 0.11 to 3.0%, and egg production decreased from 10 to 30%, with a peak mortality rate (3.0%) observed at 54 wk of age. Necropsy of 11 birds demonstrated tissue damage that included hepatitis, liver hemorrhage, rupture, and/or enlarged livers. Microscopic liver lesions exhibited hepatocytic necrosis, lymphocytic periphlebitis, and myeloid leukosis. While no bacteria were recovered from liver and spleen samples, avian hepatitis E virus (HEV) RNA was detected in all 11 tested hens by nested reverse transcription-polymerase chain reaction. Of these, subgroup J avian leukosis virus (ALV-J) proviral DNA was detected in 5 hens by PCR. Alignments of partial ORF2 gene sequences obtained here demonstrated shared identity (76 to 97%) with corresponding sequences of other known avian HEV isolates. Env sequences of ALV-J isolates obtained here shared 50.1 to 55% identity with other ALV subgroups and 91.8 to 95.5% identity with other known ALV-J isolates. Phylogenetic tree analysis of selected sequences obtained here grouped an avian HEV sequence with genotype 3 HEV and assigned an ALV-J sequence to a branch separate from known ALV-J subgroups. Immunohistochemical results confirmed the presence of avian HEV and ALV-J in livers. Therefore, these results suggest that avian HEV and ALV-J co-infection caused the outbreak of hepatitis and liver hemorrhagic syndrome observed in the layer hen flock analyzed in this study.

Effects of PRRSV Infection on the Porcine Thymus.

Porcine reproductive and respiratory syndrome virus (PRRSV) dramatically affects the thymus and its ability to carry out its normal functions. In particular, infection incapacitates PRRSV-susceptible CD14pos antigen-presenting cells (APCs) in the thymus and throughout the body. PRRSV-induced autophagy in thymic epithelial cells modulates the development of T cells, and PRRSV-induced apoptosis in CD4posCD8pos thymocytes modulates cellular immunity against PRRSV and other pathogens. Pigs are less able to resist and/or eliminate secondary infectious agents due the effect of PRRSV on the thymus, and this susceptibility phenomenon is long recognized as a primary characteristic of PRRSV infection.

Direct Interaction Between CD163 N-Terminal Domain and MYH9 C-Terminal Domain Contributes to Porcine Reproductive and Respiratory Syndrome Virus Internalization by Permissive Cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) has a highly restricted tropism for cells of the monocyte-macrophage lineage, including porcine alveolar macrophages (PAMs). PRRSV entry into permissive cells involves several mediators in addition to two required host cell receptors, CD163 and MYH9. It is unknown whether CD163 directly interacts and/or cooperates with MYH9 to facilitate PRRSV infection. In this study, CD163 and MYH9 were co-immunoprecipitated from PAMs regardless of PRRSV infection status. Further truncation analysis indicated that the CD163 N-terminal region, containing scavenger receptor cysteine-rich domains 1 to 4 (SRCR1-4), directly interacts with the MYH9 C-terminal domain region without involvement of other adaptor proteins. Meanwhile, non-permissive HEK293T cells that stably expressed truncated swine CD163 SRCR1-4 domain did not support virus attachment. However, virus attachment to cells stably expressing SRCR5-CT domain was demonstrated to occur without appreciable virus internalization. The involvement of the SRCR1-4 domain in virus internalization was further demonstrated by the fact that incubation of recombinant SRCR1-4 protein with PAMs abolished subsequent virus internalization by permissive cells. These results demonstrated that CD163 SRCR1-4 interacts with the MYH9 C-terminal domain to facilitate PRRSV virion internalization in permissive cells, thus expanding our understanding of PRRSV cell-invasion mechanisms.

Thermus caldilimi sp. nov., a thermophilic bacterium isolated from a geothermal area.

A Gram-stain negative, aerobic bacterium, designated strain YIM 78456T, was isolated from a hot spring sediment, Ngamring county, Tibet, south-west China. The taxonomic position of the isolate was investigated by a polyphasic approach. The novel isolate was found to be aerobic and rod-shaped. Colonies were observed to be pale yellow and circular. The strain was found to grow at pH 7.0-8.0 (optimum, pH 7.0), 45-65 °C (optimum, 55 °C) and in the presence of up to 1.5% NaCl. Comparison of the 16S rRNA gene sequence of strain YIM 78456T and other members of the genus Thermus showed sequence similarities ranging from 90.3 to 97.3%, with strain YIM 78456T showing close sequence similarity to Thermus caliditerrae YIM 77925T (97.3%). The phylogenetic trees based on 16S rRNA gene sequences showed that strain YIM 78456T forms a distinct clade with T. caliditerrae YIM 77925T. The predominant menaquinone was identified as MK-8 and the DNA G+C content was determined to be 65.1 mol%. The major cellular fatty acids (> 10%) were identified as iso-C15:0, anteiso-C15:0 and iso-C17:0. The polar lipids were found to consist of an aminophospholipid, a phospholipid and glycolipids. On the basis of the morphological and chemotaxonomic characteristics, as well as genotypic data, it is proposed that strain YIM 78456T represents a novel species of the genus Thermus, for which the name Thermus caldilimi sp. nov. is proposed. The type strain is YIM 78456T (= KCTC 52948T = NBRC 113036T).

Development of a monoclonal antibody against swine leukocyte antigen (SLA)-DR α chain and evaluation of SLA-DR expression in bone marrow-derived dendritic cells after PRRSV infection.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most common diseases in the global swine industry. PRRSV infection is highly restricted to cells of the monocyte-macrophage lineage. However, the lack of antibodies to swine monocyte-macrophage lineage markers significantly hampers PRRSV research. In this study, we have developed a monoclonal antibody against the swine leukocyte antigen (SLA)-DRα chain and confirmed its reactivity with endogenous expressed SLA-DR in a variety of cell lines and primary swine antigen-presenting cells (PAMs, PBMC and BM-DCs). Moreover, the level of SLA-DR expression after PRRSV infection were evaluated by our homemade Mab and a commercial anti-SLA-DR antibody. Based on our result, the protein level of SLA-DRα expression is increased after PRRSV infection in DC, while the mRNA of both SLA-DRα and SLA-DRß were significantly inhibited by PRRSV replication. In conclusion, we successfully developed a MAb reactive with endogenous SLA-DR in western blotting, and this MAb could be a useful tool for further research and analysis. Moreover, the inconsistency of SLA-DR expression between protein and mRNA levels may suggest a novel role of DC played during the immune response after PRRSV infection.

Novosphingobium meiothermophilum sp. nov., isolated from a hot spring.

A moderately thermophilic, aerobic, Gram-stain-negative, non-spore-forming, rod-shaped and yellow-pigmented bacterium, designated strain SYSU G00007T, was isolated from a hot spring slurry sample. Optimum growth was observed at 37-45 °C and pH 7. Pairwise comparison of the 16S rRNA gene sequence of strain SYSU G00007T and other Novosphingobium species showed sequence similarities ranging from 93.7 to 97.9 %. Strain SYSU G00007T showed highest sequence identity to Novosphingobium subterraneum DSM 12447T (97.9 %). The average nucleotide identities and digital DNA-DNA hybridization values between strain SYSU G00007T and its closely related phylogenetic neighbours were below 81 and 31 %, respectively, indicating that strain SYSU G00007T represented a novel species of the genus Novosphingobium. The DNA G+C content of strain SYSU G00007T was 64.3 % (genome). The major respiratory quinone was ubiquinone Q-10. The polar lipid profile included diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, two sphingoglycolipids, two unidentified phospholipids, two unidentified aminophospholipids and two unidentified polar lipids. Spermidine was the only polyamine detected. The major fatty acids were C19 : 0cyclo ω8c, summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) and C16 : 0. The results obtained from phylogenetic, chemotaxonomic and phenotypic analyses support the conclusion that strain SYSU G00007T represents a novel species of the genus Novosphingobium, for which we proposed the name Novosphingobiummeiothermophilum sp. nov. The type strain is SYSU G00007T (=KCTC 52672T=CCTCC AB2017010T).

Chicken Organic Anion-Transporting Polypeptide 1A2, a Novel Avian Hepatitis E Virus (HEV) ORF2-Interacting Protein, Is Involved in Avian HEV Infection.

Avian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease in chickens. Due to the absence of a highly effective cell culture system, there are few reports about the interaction between avian HEV and host cells. In this study, organic anion-transporting polypeptide 1A2 (OATP1A2) from chicken liver cells was identified to interact with ap237, a truncated avian HEV capsid protein spanning amino acids 313 to 549, by a glutathione S-transferase (GST) pulldown assay. GST pulldown and indirect enzyme-linked immunosorbent assays (ELISAs) further confirmed that the extracellular domain of OATP1A2 directly binds with ap237. The expression levels of OATP1A2 in host cells are positively correlated with the amounts of ap237 attachment and virus infection. The distribution of OATP1A2 in different tissues is consistent with avian HEV infection in vivo Finally, when the functions of OATP1A2 in cells are inhibited by its substrates or an inhibitor or blocked by ap237 or anti-OATP1A2 sera, attachment to and infection of host cells by avian HEV are significantly reduced. Collectively, these results displayed for the first time that OATP1A2 interacts with the avian HEV capsid protein and can influence viral infection in host cells. The present study provides new insight to understand the process of avian HEV infection of host cells.IMPORTANCE The process of viral infection is centered around the interaction between the virus and host cells. Due to the lack of a highly effective cell culture system in vitro, there is little understanding about the interaction between avian HEV and its host cells. In this study, a total of seven host proteins were screened in chicken liver cells by a truncated avian HEV capsid protein (ap237) in which the host protein OATP1A2 interacted with ap237. Overexpression of OATP1A2 in the cells can promote ap237 adsorption as well as avian HEV adsorption and infection of the cells. When the function of OATP1A2 in cells was inhibited by substrates or inhibitors, attachment and infection by avian HEV significantly decreased. The distribution of OATP1A2 in different chicken tissues corresponded with that in tissues during avian HEV infection. This is the first finding that OATP1A2 is involved in viral infection of host cells.

Calidifontimicrobium sediminis gen. nov., sp. nov., a new member of the family Comamonadaceae.

Two Gram-stain-negative and facultative anaerobic bacteria, designated strains SYSU G00088T and YIM 73032, were isolated from sediment samples collected from hot springs in Tibet, China. Based on the analyses of the 16S rRNA gene sequences, the two isolates were observed to be a member of the family Comamonadaceae, sharing highest pairwise sequence identity with type strains of Piscinibacter defluvii SH-1T (97.8  and 97.7 %, respectively). They were able to grow in the temperature range of 37-50 °C, pH 5.0-9.0 and in the presence of up to 0.5 % (w/v) NaCl. Both strains were positive for catalase and oxidase reactions. Cells of the novel isolates were short-rods and motile by means of a polar flagellum. The chemotaxonomic features of the two strains include ubiquinone 8 as the respiratory isoprenologue, diphosphatidylglycerol, phosphatidylethanolamine and phoshatidylglycerol as the known polar lipids, and C16 : 0 and C17 : 0cyclo as major fatty acids. The genomic DNA of strains SYSU G00088T and YIM 73032 had G+C contents of 71.8 and 71.9%, respectively. Based on the analyses of the phylogenetic, chemotaxonomic, morphological and genomic data, the two isolates are considered to represent a novel species of a new genus, for which the name Calidifontimicrobium sediminis gen. nov., sp. nov. is proposed. The type strain of Calidifontimicrobium sediminis is SYSU G00088T (=KCTC 52671T=CGMCC 1.13597T).