Establishment of a TaqMan probe-based qPCR assay for detecting microsporidia Enterospora epinepheli in grouper.

Enterospora epinepheli is an intranuclear microsporidian parasite causing serious emaciative disease in hatchery-bred juvenile groupers (Epinephelus spp.). Rapid and sensitive detection is urgently needed as its chronic infection tends to cause emaciation as well as white faeces syndrome and results in fry mortality. This study established a TaqMan probe-based real-time quantitative PCR assays targeting the small subunit rRNA (SSU) gene of E. epinepheli. The relationship between the standard curve of cycle threshold (Ct) and the logarithmic starting quantity (SQ) was determined as Ct = -3.177 lg (SQ) + 38.397. The correlation coefficient (R2 ) was 0.999, and the amplification efficiency was 106.4%. The detection limit of the TaqMan probe-based qPCR assay was 1.0 × 101 copies/µL and that is 100 times sensitive than the traditional PCR method. There is no cross-reaction with other aquatic microsporidia such as Ecytonucleospora hepatopenaei, Nucleospora hippocampi, Potaspora sp., Ameson portunus. The intra-assay and inter-assay showed great repeatability and reproducibility. In addition, the test of clinical samples showed that this assay effectively detected E. epinepheli in the grouper's intestine tissue. The established TaqMan qPCR assays will be a valuable diagnostic tool for the epidemiological investigation as well as prevention and control of E. epinepheli.

Microsporidia infection upregulates host energy metabolism but maintains ATP homeostasis.

Microsporidia are a group of obligate intracellular parasites which lack mitochondria and have highly reduced genomes. Therefore, they are unable to produce ATP via the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. Instead, they have evolved strategies to obtain and manipulate host metabolism to acquire nutrients. However, little is known about how microsporidia modulate host energy metabolisms. Here, we present the first targeted metabolomics study to investigate changes in host energy metabolism as a result of infection by a microsporidian. Metabolites of silkworm embryo cell (BmE) were measured 48 h post infection by Nosema bombycis. Thirty metabolites were detected, nine of which were upregulated and mainly involved in glycolysis (glucose 6-phosphate, fructose 1,6-bisphosphate) and the TCA cycle (succinate, α-ketoglutarate, cis-aconitate, isocitrate, citrate, fumarate). Pathway enrichment analysis suggested that the upregulated metabolites could promote the synthesization of nucleotides, fatty acids, and amino acids by the host. ATP concentration in host cells, however, was not significantly changed by the infection. This ATP homeostasis was also found in Encephalitozoon hellem infected mouse macrophage RAW264.7, human monocytic leukemia THP-1, human embryonic kidney 293, and human foreskin fibroblast cells. These findings suggest that microsporidia have evolved strategies to maintain levels of ATP in the host while stimulating metabolic pathways to provide additional nutrients for the parasite.

Characterization of Hsp70 gene family provides insight into its functions related to microsporidian proliferation.

Heat shock protein 70 (Hsp70), a highly conserved protein family, is widely distributed in organisms and plays fundamental roles in biotic and abiotic stress responses. However, reports on Hsp70 genes are scarce in microsporidia, a very large group of obligate intracellular parasites that can infect nearly all animals, including humans. In this study, we identified 37 Hsp70 proteins from eight microsporidian genomes and classified them into four subfamilies (A-D). The number of Hsp70 genes in these microsporidia was significantly fewer than in Rozella allomycis and yeast. All microsporidian species contained genes from each subfamily and similar subcellular locations (mitochondria, endoplasmic reticulum, cytosol, and cytosol and/or nucleus), indicating that each Hsp70 member may fulfil distinct functions. The conserved structures and motifs of the Hsp70 proteins in the same subfamily were highly similar. Expression analysis indicated that the subfamily C cytosol (cyto)-associated Hsp70s is functional during microsporidia development. Immunofluorescence assays revealed that Cyto-NbHsp70 was cytoplasmically located in the proliferation-stage of Nosema bombycis. Cyto-NbHsp70 antiserum also labeled Encephalitozoon hellem within infected cells, suggesting that this antiserum is a potential molecular marker for labeling the proliferative phases of different microsporidian species. The propagation of N. bombycis was significantly inhibited following RNAi of Cyto-NbHsp70, indicating that Cyto-NbHsp70 is important for pathogen proliferation. Our phylogenetic data suggest that Hsp70 proteins evolved during microsporidia adaption to intracellular parasitism, and they play important roles in pathogen development.

In-vitro cultivation of Nosema bombycis sporoplasms: A method for potential genetic engineering of microsporidia.

Microsporidia are obligate intracellular parasites and cannot be cultured in vitro, which limits the use of current genetic engineering technologies on this pathogen. We isolated sporoplasms of Nosema bombycis to attempt to culture the pathogen in vitro. Cell-free medium was designed and successfully maintained the sporoplasms for 5 days. The sporoplasms were able to absorb ATP from the medium and DNA replicated during cultivation, although there was not a significant change in morphology and number of sporoplasms. Our study provides a strategy for in vitro cultivation and genetic manipulation of microsporidia. .

Translocation of microbes and changes of immunocytes in the gut of rapid- and slow-progressor Chinese rhesus macaques infected with SIVmac239.

Human/simian immunodeficiency virus (HIV/SIV) infection can cause severe depletion of CD4(+) T cells in both plasma and mucosa; it also results in damage to the gut mucosa barrier, which makes the condition more conducive to microbial translocation. In this study, we used SIV-infected Chinese rhesus macaques to quantify the extent of microbial translocation and the function of immune cells in the entire gastrointestinal tract and to compare their differences between rapid and slow progressors. The results showed that in the slow progressors, microbial products translocated considerably and deeply into the lamina propria of the gut; the tissue macrophages had no significant differences compared with the rapid progressors, but there was a slightly higher percentage of mucosal CD8(+) T cells and a large amount of extracellular microbial products in the lamina propria of the intestinal mucosa of the slow progressors. The data suggested that although microbial translocation increased markedly, the mucosal macrophages and CD8(+) T cells were insufficient to clear the infiltrated microbes in the slow progressors. Also, therapies aimed at suppressing the translocation of microbial products in the mucosa could help to delay the progression of SIV disease.

Case report: Tall cell carcinoma with reversed polarity of the breast: an additional case and review of the literature.

Objective: The aim of this report was to comprehensively investigate the clinicopathological features, histological characteristics, and differential diagnosis of tall cell carcinoma with reversed polarity of the breast (TCCRP) to enhance the understanding of this tumour for precise therapeutic interventions. Methods: The clinicopathological characteristics and differential diagnosis of a patient with TCCRP were retrospectively analysed, and a systematic literature review was extracted from relevant published studies on PubMed. Results: All patients included in the study were female, with a median age of 51 years. Microscopically, the tumour cells exhibited a solid papillary growth pattern with tall columnar morphology and reversed nuclear polarity. Immunohistochemistry revealed that the tumours were triple-negative breast cancer (negative for ER, PR, and HER-2), with a low Ki-67 proliferation index. Different degrees of expression were observed for CK7, Calretinin, and S-100 markers; however, CK5/6 showed high expression levels. Conclusions: TCCRP is an uncommon invasive carcinoma subtype found in the breast. Its histological morphology resembles that of tall cell subtype papillary thyroid carcinoma. Accurate diagnosis requires the integration of histomorphological assessment along with immunohistochemistry and molecular genetics analysis.

High-throughput analytical techniques for determination of residues of 653 multiclass pesticides and chemical pollutants in tea, Part II: comparative study of extraction efficiencies of three sample preparation techniques.

This paper reports a study of the extraction efficiency for the multiresidue pesticides and chemical pollutants in tea with three methods over three stages. Method 1 adopts the Pang et al. approach: the targets were extracted with 1% acetic acid in acetonitrile and cleaned up with a Cleanert TPT SPE cartridge; Method 2 adopts the QuEChERS approach: the targets were cleaned up dispersively with graphitized carbon and primary-secondary amine (PSA) sorbent; Method 3 adopts the relatively commonly used approach of hydration for solid samples, with tea hydrated before being extracted through salting out with acetonitrile and the cleanup procedures identical to those of Method 1. The three stages comprised two phases of comparative tests on spike recoveries of 201 pesticides and chemical pollutants from different teas and a third phase on determination of the content of the 201 pesticides and chemical pollutants from aged tea samples. In stages I and II, test results of the spike recoveries of 201 pesticides and chemical pollutants demonstrated that 91.4% of the pesticide and chemical pollutant recoveries fell within the range of 70-110%, and 93.2% of the pesticides and chemical pollutants had RSD < 15%, with no marked difference obtained by Method 1 and Method 2 regardless of whether it was green tea or woolong tea, or GC/MS or GC/MS/MS was used for analysis. For pigment removal, Method 1 was superior to Method 2; in terms of easy operation, Method 2 outweighed Method 1. However, Method 3 obtained relatively low recoveries, with 94% of pesticide and chemical pollutant recoveries less than 70%, which proved that Method 3 was not applicable to the determination of multiresidue pesticides and chemical pollutants in tea. Stage III made a comparison of Method 1 and Method 2 for the extraction efficiency of pesticides and chemical pollutants in 165-day-aged samples of green and woolong tea. Test results showed that 94% of the pesticide and chemical pollutant content in the aged tea samples was recovered with Method 1, more than 10% higher than with Method 2 (30-50% higher on average). For green tea, 193 (GC/MS/MS) and 197 (GC/MS) pesticides and chemical pollutants accounted for 96.5% (GC/MS/MS) and 98.0% (GC/MS) with Method 1 higher than with Method 2. For woolong tea, 191 (GC/MS/MS) and 194 (GC/MS) pesticides and chemical pollutants accounted for 95% (GC/MS/MS) and 96% (GC/MS/MS) with Method 1, higher than with Method 2, respectively. In other words, there were definite differences in the test results for aged tea samples between Method 1 and Method 2, which suggests that Method 1 was capable of extracting more residual pesticides and chemical pollutants from the precipitated 165-day-aged tea samples. The reason can be traced to the possibility that Method 1 (high-speed homogenizing) has better extraction efficiency than Method 2 (vortex and oscillation). Therefore, Method 1 was chosen as the sample preparation technique for multiresidue pesticide and chemical pollutant analysis in tea.

MCM6 is a Poor Prognostic Biomarker and Promotes Progression in Breast Cancer.

BACKGROUND: Breast cancer is the commonest global malignancy and the primary cause of carcinoma death. MCM6 is vital to carcinogenesis, but the pathogenesis of MCM6 remains unclear. METHODS: MCM6 expression in patients with breast cancer was examined through The Cancer Genome Atlas (TCGA) database, immunohistochemistry, Quantitative Real-Time PCR (qRT‒PCR) and Western blotting. The prognostic factors were assessed by the Kaplan‒Meier method and Cox regression. On the basis of the key factors selected by multivariable Cox regression analysis, a nomogram risk prediction model was adopted for clinical risk assessment. The TCGA database was utilized to determine how MCM6 is correlated with chemotherapy sensitivity, immune checkpoint-related genes (ICGs), tumor-infiltrating immune cells, along with tumor mutation burden (TMB) and methylation. The impact of MCM6 on carcinoma cells was investigated in terms of proliferation, cell cycle as well as migrating and invasive behavior through CCK assays, flow cytometry, wound healing assays, Transwell assays and xenotransplantation experiments. RESULTS: MCM6 expression was upregulated, which is closely associated with the size of the tumor (p = 0.001) and lymph node metastasis (p = 0.012) in patients with breast cancer. Multivariate analysis revealed MCM6 to be an independent risk factor for prognosis in patients with breast carcinoma. The nomograph prediction model included MCM6, age, ER, M and N stage, which displayed good discrimination with a C index of 0.817 and good calibration. Overexpression of MCM6 correlated with chemotherapy sensitivity, immune checkpoint-related genes (ICGs), tumor-infiltrating immune cells, tumor mutation burden (TMB), and methylation. Silencing MCM6 significantly inhibited proliferation, prolonged the G1 phase of the cell cycle, and restrained the proliferation, migration and invasive behavior of cancerous cells and inhibited tumor growth in vivo. CONCLUSIONS: Our research shows that MCM6 is highly expressed in breast cancer and can be used as an independent prognostic factor, which is expected to become a new target for the treatment of breast cancer in the future.

Case Report: Clinicopathological Analysis of Minute Pulmonary Meningothelial-Like Nodules: Report of 7 Cases.

Objective: To investigate the clinical manifestations, radiologic features, pathological features, and immunophenotype of minute pulmonary meningothelial-like nodules (MPMNs). Method: This is a retrospective observational study. We collected the clinical data of 7 cases of MPMNs, and performed comprehensive characterization using a combination of clinical, morphological, radiologic and immunohistochemical assessments. Results: Of the 7 cases of MPMNs, 6 were female and 1 was male. The median age was 55 years. All MPMNs were multiple in lung with the size from 0,01 to 0,5cm. Chest CT examination showed ground-glass attenuation or solid nodules. Four cases were concomitant with carcinoma and/or pneumonia, and 3 cases occurred alone. Four of the 7 patients had no obvious symptoms; 3 patients had chest pain or cough or shortness of breath or hemoptysis. Multiple white nodules were found macroscopically, and the diseased cells grew along the alveolar septum, with relatively normal morphology, rich cytoplasm, unclear cell boundary, and uniform nucleus with delicate chromatin and without atypia; and the diseased cells showed nest or whorls distribution. EMA, PR, CD56 and vimentin were positive in all cases by immunohistochemistry. Conclusions: MPMNs are rare benign lesions in the lung, often multiple, usually less than 0.5cm in diameter, most of which have no obvious clinical symptoms. MPMNs are often found by chest CT, and occur independently or concomitant with other lesions. The positive immunohistochemical staining of EMA, PR, CD56, vimentin supports the diagnosis.

In vivo study of dual functionalized mussel-derived bioactive peptides promoting 3D-printed porous Ti6Al4V scaffolds for repair of rabbit femoral defects.

The 3D printed porous titanium alloy scaffolds are beneficial to enhance angiogenesis, osteoblast adhesion, and promote osseointegration. However, titanium alloys are biologically inert, which makes the bond between the implant and bone tissue weak and prone to loosening. Inspired by the natural biological marine mussels, we designed four-claw-shaped mussel-derived bioactive peptides for the decoration of porous titanium alloy scaffolds: adhesion peptide-DOPA, anchoring peptide-RGD and osteogenic-inducing peptide-BMP-2. And the bifunctionalization of 3D-printed porous titanium alloy scaffolds was evaluated in vivo in a rabbit model of bone defect with excellent promotion of osseointegration and mechanical stability. Our results show that the in vivo osseointegration ability of the modified 3D printed porous titanium alloy test piece is significantly improved, and the bifunctional polypeptide coating group E has the strongest osseointegration ability. In conclusion, our experimental design partially solves the problems of stress shielding effect and biological inertness, and provides a convenient and feasible method for the clinical application of titanium alloy implants in biomedical implant materials.

Reduced stratifin expression can serve as an independent prognostic factor for poor survival in patients with esophageal squamous cell carcinoma.

UNLABELLED: Stratifin plays an important role in cancer biology by interfering with intracellular signalling pathways and cell-cycle checkpoints. Decreased expression of stratifin gene has been reported to be a poor prognostic indicator in a variety of human malignant tumors. AIM: To clarify the role and prognostic significance of stratifin in esophageal squamous cell carcinoma (ESCC). METHODS: The alteration of stratifin messenger RNA (mRNA) and protein was analyzed by reverse-transcription and quantitative real-time polymerase chain reaction (QRT-PCR) and Western blotting in 20 paired ESCC and nonneoplastic esophageal mucosa tissues, respectively. Then, immunohistochemistry (IHC) was used to evaluate expression of stratifin in tissues of 148 ESCC patients (including the former 20 pairs of tissues) and correlate it with clinicopathological parameters and prognosis of ESCC patients. RESULTS: The stratifin level of mRNA and protein was markedly downregulated in ESCC tissue compared with in corresponding nonneoplastic esophageal epithelium (P<0.05). Similarly, the positive rate of stratifin protein expression was lower in the esophageal cancer than in paired nonneoplastic esophageal epithelium as detected by IHC (P=0.007). Statistically, the downregulation of stratifin expression was correlated with tumor infiltration depth (P=0.003), lymph node metastasis (P=0.008), distant metastasis (P=0.013), and lymphovascular invasion (P=0.007) of ESCC. Furthermore, the reduced stratifin expression was associated with shorter 5-year survival rate of ESCC patients after curative surgery (P<0.0001). On the basis of univariate and multivariate Cox regression analysis, we found that reduced stratifin expression, T4 stage, lymph node metastasis, and distant metastasis were independent risk factors for worse prognosis in ESCC patients. CONCLUSION: The present report indicates that stratifin could be a useful indicator for prognosis of this disease, as well as a potential target for more effective therapy.

Morphology and Transcriptome Analysis of Nosema bombycis Sporoplasm and Insights into the Initial Infection of Microsporidia.

Microsporidia are obligate intracellular parasites that infect a wide variety of host organisms, including humans. The sporoplasm is the initial stage of microsporidian infection and proliferation, but its morphological and molecular characteristics are poorly understood. In this study, the sporoplasm of Nosema bombycis was successfully isolated and characterized after the induction of spore germination in vitro The sporoplasm was spherical, 3.64 ± 0.41 µm in diameter, had the typical two nuclei, and was nonrefractive. Scanning and transmission electron microscopy analyses revealed that the sporoplasm was surrounded by a single membrane, and the cytoplasm was usually filled with relatively homogeneous granules, possibly ribosomes, and contained a vesicular structure comprising a concentric ring and coiled tubules. Propidium iodide staining revealed that the sporoplasm membrane showed stronger membrane permeability than did the cell plasma membrane. Transmission electron microscopy (TEM) revealed that the sporoplasm can gain entry to the host cell by phagocytosis. Transcriptome analysis of mature spores and sporoplasms showed that 541 significantly differentially expressed genes were screened (adjusted P value [Padj] < 0.05), of which 302 genes were upregulated and 239 genes were downregulated in the sporoplasm. The majority of the genes involved in trehalose synthesis metabolism, glycolysis, and the pentose phosphate pathway were downregulated, whereas 10 transporter genes were upregulated, suggesting that the sporoplasm may inhibit its own carbon metabolic activity and obtain the substances required for proliferation through transporter proteins. This study represents the first comprehensive and in-depth investigation of the sporoplasm at the morphological and molecular levels and provides novel insights into the biology of microsporidia and their infection mechanism.IMPORTANCE Once awoken from dormancy, the cellular matter of microsporidia is delivered directly into the host cell cytoplasm through the polar tube. This means that the microsporidia are difficult to study biologically in their active state without a contaminating signal from the host cell. Sporoplasm is a cell type of microsporidia in vitro, but relatively little attention has been paid to the sporoplasm in the past 150 years due to a lack of an effective separation method. Nosema bombycis, the first reported microsporidium, is a type of obligate intracellular parasite that infects silkworms and can be induced to germinate in alkaline solution in vitro We successfully separated the N. bombycis sporoplasm in vitro, and the morphological and structural characteristics were investigated. These results provide important insight into the biology and pathogenesis of microsporidia and potentially provide a possible strategy for genetic manipulation of microsporidia targeting the sporoplasm.

Evolutionary and functional studies on microsporidian ATP-binding cassettes: Insights into the adaptation of microsporidia to obligated intracellular parasitism.

ATP-binding cassette (ABC) transporters comprise the largest family of transmembrane proteins and are found in all domains of life. The ABCs are involved in a variety of biological processes and as exporters play important roles in multidrug resistance. However, the ABC transporters have not been addressed in microsporidia, which are a very large group of obligate intracellular parasites that can infect nearly all animals, including humans. Here, a total of 234 ABC transporters were identified from 18 microsporidian genomes and classified into five subfamilies, including 74 ABCBs, 2 ABCCs, 18 ABCEs, 15 ABCFs, 102 ABCGs and 23 uncategorized members. Two subfamilies, ABCA and ABCD, are found in most organisms, but lost in microsporidia. Phylogenetic analysis indicated that microsporidian ABCB and ABCG subfamilies expanded by recent gene duplications, which resulted in the two largest subfamilies in microsporidia. Functional analysis via qRT-PCR and Western blotting revealed that NoboABCG1.1, an ABCG member of Nosema bombycis, is expressed in mature spores and up-regulated from 1 dpi to 6 dpi in infected silkworm midgut. IFA and IEM analysis showed that NoboABCG1.1 is localized on the plasma membrane of the sporoplasm, meront and mature spore. The propagation of N. bombycis was significantly inhibited after the RNAi of NoboABCG1.1 expression, indicating that NoboABCG1.1 is important to the pathogen proliferation. In conclusion, our study uncovered that the ABCs evolved during microsporidia adaption to intracellular parasitism and play important roles in the pathogen development.

SilkPathDB: a comprehensive resource for the study of silkworm pathogens.

Silkworm pathogens have been heavily impeding the development of sericultural industry and play important roles in lepidopteran ecology, and some of which are used as biological insecticides. Rapid advances in studies on the omics of silkworm pathogens have produced a large amount of data, which need to be brought together centrally in a coherent and systematic manner. This will facilitate the reuse of these data for further analysis. We have collected genomic data for 86 silkworm pathogens from 4 taxa (fungi, microsporidia, bacteria and viruses) and from 4 lepidopteran hosts, and developed the open-access Silkworm Pathogen Database (SilkPathDB) to make this information readily available. The implementation of SilkPathDB involves integrating Drupal and GBrowse as a graphic interface for a Chado relational database which houses all of the datasets involved. The genomes have been assembled and annotated for comparative purposes and allow the search and analysis of homologous sequences, transposable elements, protein subcellular locations, including secreted proteins, and gene ontology. We believe that the SilkPathDB will aid researchers in the identification of silkworm parasites, understanding the mechanisms of silkworm infections, and the developmental ecology of silkworm parasites (gene expression) and their hosts. Database URL: http://silkpathdb.swu.edu.cn.

Increased expression of prohibitin and its relationship with poor prognosis in esophageal squamous cell carcinoma.

Prohibitin, a potential tumor suppressor, has been shown to be an anti- proliferative protein, a regulator of cell-cycle progression and in apoptosis. Recently, it was found to be over-expressed in breast cancer and gastric cancer, and it has been suggested as a biomarker in those diseases. To clarify the role and the prognostic significance of prohibitin expression in esophageal squamous cell carcinoma (ESCC), we analyzed the expression in ESCC and their corresponding nonneoplastic epithelia tissues by immunohistochemistry(IHC), Western blotting and real-time quantitative reverse transcription polymerase chain reaction(QRT-PCR).The relationship between prohibitin expression and clinicopathological variables was examined by statistical analysis. The findings suggested the up-regulation of prohibitin play an important role in the carcinogenesis of ESCC. The over-expression of prihibitin was significantly correlated with the depth of tumor, lymph node metastasis, distant metastasis, lymphatic invasion and vascular invasion of ESCC. These results suggested that prohibitin(+), lymph node metastasis and distant metastasis could be the independent risk factors for worse prognosis in ESCC patients.

Expression of semaphorin 5A and its receptor plexin B3 contributes to invasion and metastasis of gastric carcinoma.

AIM: To investigate the protein and mRNA expression of semaphorin 5A and its receptor plexin B3 in gastric carcinoma and explore its role in the invasion and metastasis of gastric carcinoma. METHODS: Expression of semaphorin 5A and its receptor plexin B3 in 48 samples of primary gastric carcinoma, its corresponding non-neoplastic mucosa, and matched regional lymph node metastasis was assayed by reverse transcription-polymerase chain reaction (RT-PCR), real-time RT-PCR and Western blotting. RESULTS: The protein and mRNA expression of semaphorin 5A and its receptor plexin B3 increased gradually in non-neoplastic mucosa, primary gastric carcinoma and lymph node metastasis (P < 0.05). Moreover, the expression of semaphorin 5A was closely correlated with that of plexin B3. CONCLUSION: Semaphorin 5A and its receptor plexin B3 play an important role in the invasion and metastasis of gastric carcinoma.

[The analysis of bro genes of GD isolate of Bombyx mori nucleopolyhedrovirus].

BmNPV GD isolate from China was plaque-purified and four bro genes were cloned termed as bro-a,b,c,d. The obtained sequences were aligned to the related sequences in GenBank and the BmNPV CQ1 isolate preserved in our laboratory. Compared with genome sequences of BmNPV T3 isolate, bro genes of GD isolate housed insertion and deletion, and the changes of amino acid mainly occured at the N terminal of corresponding protein. The phylogenetic analysis of bro genes indicated that GD bro-d gene belongs to subgroup A together with T3, CQ1 bro-d and SC7 bro- III; GD bro-a, c genes belong to subgroup B together with T3, CQ1 bro-a, c and SC7 bro-II; GD bro-b gene belongs to subgroup C together with T3, CQ1 bro-b, e and SC7 bro-I. The evolutionary relationship of bro genes showed vague relevance to their geographical location. The distribution character of bro genes in four BmNPV isolates is coincidence with the KANG's theory that the bro-d plays an irreplaceable functional role(s) during viral replication, while bro-a and bro-c functionally complement each other. Meanwhile, we postulate that 3 bro genes in SC7 isolate is probable the most simple form of bro genes.