Bioproduction and immunogenic evaluation of SARS-CoV-2 prototype vaccine in silkworm BmN cells.

COVID-19, caused by the novel coronavirus SARS-CoV-2, has presented a significant challenge to global health, security, and the economy. Vaccination is considered a crucial measure in preventing virus transmission. The silkworm bioreactor has gained widespread usage in antigen presentation, monoclonal antibody preparation, and subunit vaccine development due to its safety, efficiency, convenience, and cost-effectiveness. In this study, we employed silkworm BmN cells and the silkworm MultiBac multigene co-expression system to successfully produce two prototype vaccines: a recombinant baculovirus vector vaccine (NPV) co-displaying the SARS-CoV-2 virus capsid protein and a capsid protein virus-like particle (VLP) vaccine. Following the purification of these vaccines, we immunized BALB/c mice to evaluate their immunogenicity. Our results demonstrated that both VLP and NPV prototype vaccines effectively elicited robust immune responses in mice. However, when equal inoculation doses between groups were compared, the recombinant NPV vaccine exhibited significantly higher serum antibody titers and increased expression of spleen cytokines and lymphocyte immune regulatory factors compared to the VLP group. These results suggested an increased immune efficacy of the recombinant NPV vaccine. Conversely, the VLP prototype vaccine displayed more pronounced effects on lymphocyte cell differentiation induction. This study successfully constructed two distinct morphological recombinant vaccine models and systematically elucidated their differences in humoral immune response and lymphocyte differentiation rate. Furthermore, it has fully harnessed the immense potential of silkworm bioreactors for vaccine research and development, providing valuable technical insights for studying mutated viruses like coronaviruses.

Impact of the Transboundary Interference Inhibitor on RNAi and the Baculovirus Expression System in Insect Cells.

RNA interference inhibitors were initially discovered in plant viruses, representing a unique mechanism employed by these viruses to counteract host RNA interference. This mechanism has found extensive applications in plant disease resistance breeding and other fields; however, the impact of such interference inhibitors on insect cell RNA interference remains largely unknown. In this study, we screened three distinct interference inhibitors from plant and mammal viruses that act through different mechanisms and systematically investigated their effects on the insect cell cycle and baculovirus infection period at various time intervals. Our findings demonstrated that the viral suppressors of RNA silencing (VSRs) derived from plant and mammal viruses significantly attenuated the RNA interference effect in insect cells, as evidenced by reduced apoptosis rates, altered gene regulation patterns in cells, enhanced expression of exogenous proteins, and improved production efficiency of recombinant virus progeny. Further investigations revealed that the early expression of VSRs yielded superior results compared with late expression during RNA interference processes. Additionally, our results indicated that dsRNA-binding inhibition exhibited more pronounced effects than other modes of action employed by these interference inhibitors. The outcomes presented herein provide novel insights into enhancing defense mechanisms within insect cells using plant and mammal single-stranded RNA virus-derived interference inhibitors and have potential implications for expanding the scope of transformation within insect cell expression systems.

Seroepidemiology and associated risk factors of brucellosis in small ruminants of district Khanewal, Pakistan.

Objectives: Keeping in view the economic and veterinary public health importance of brucellosis, this research was conducted to determine its seroprevalence and associated risk determinants in small ruminants in district Khanewal, Southern Punjab, Pakistan. Materials and Methods: Two-stage cluster sampling technique was used for sampling, and the sample size was calculated using C-survey 2.0. Accordingly, sera samples (n = 392) were collected from small ruminants in the study area from October 2022 to July 2023. All the samples were tested for the presence of anti-Brucella antibodies by Rose Bengal Plate Test (RBPT), followed by confirmation of all the samples using an enzyme linked immunosorbent assay (ELISA) kit (ID.vet®, France; sensitivity and specificity=100%, each). Results: The seropositivity rate of brucellosis was 7.14% [n = 28/392; 95% confidence interval (CI) = 4.87%-10.12%] by RBPT, whereas the results of ELISA showed an overall seroprevalence rate of 7.40% (n = 29/392; 95% CI = 5.11%-10.37%) in the study population. Univariate analysis of risk factors revealed that abortion history (AH), retained fetal membranes (RFMs), repeat breeding, flock size (FS), educational status of farmers (ESFs), awareness about brucellosis (AB), and farm hygiene had a significant association with the seroprevalence of brucellosis (p < 0.05). The multivariate analysis using a binary logistic regression model revealed that variables including tehsil, FS, AH, RFM, ESF, AB, and farming system were significant factors (p < 0.05) associated with brucellosis in the target population. Conclusion: Brucellosis is prevalent in small ruminants in Khanewal, Pakistan. The disease burden can be reduced by improving the reproductive health of animals, farm hygiene, and farmers' awareness about the diseases. Further studies are needed on a larger scale to devise stringent disease control strategies to avoid losses associated with brucellosis at regional, national, and global levels.

Systematic analysis of innate immune-related genes in the silkworm: Application to antiviral research.

The silkworm, a crucial model organism of the Lepidoptera, offers an excellent platform for investigating the molecular mechanisms underlying the innate immune response of insects toward pathogens. Over the years, researchers worldwide have identified numerous immune-related genes in silkworms. However, these identified silkworm immune genes are not well classified and not well known to the scientific community. With the availability of the latest genome data of silkworms and the extensive research on silkworm immunity, it has become imperative to systematically categorize the immune genes of silkworms with different database IDs. In this study, we present a meticulous organization of prevalent immune-related genes in the domestic silkworm, using the SilkDB 3.0 database as a reliable source for updated gene information. Furthermore, utilizing the available data, we classify the collected immune genes into distinct categories: pattern recognition receptors, classical immune pathways, effector genes and others. In-depth data analysis has enabled us to predict some potential antiviral genes. Subsequently, we performed antiviral experiments on selected genes, exploring their impact on Bombyx mori nucleopolyhedrovirus replication. The outcomes of this research furnish novel insights into the immune genes of the silkworm, consequently fostering advancements in the field of silkworm immunity research by establishing a comprehensive classification and functional understanding of immune-related genes in the silkworm. This study contributes to the broader understanding of insect immune responses and opens up new avenues for future investigations in the domain of host-pathogen interactions.

Epidemiology of Brucellosis in Small Ruminants of Rural and Peri-Urban Areas of Multan, Pakistan.

Brucellosis is a widespread zoonotic disease of veterinary and public health importance with considerably higher prevalence in developing/underdeveloped countries. This study reports the prevalence and risk determinants of brucellosis in small ruminants of peri-urban and rural areas of district Multan, Southern Punjab, Pakistan. For this purpose, sera samples (n = 392) of small ruminants were collected and subjected to preliminary screening using commercially available RBPT reagents followed by serodetection of brucellosis using multispecies i-ELISA kit (ID.vet, France). All the ELISA positive samples were confirmed by PCR using genus-specific primers, and frequencies of Brucella species in positive samples were enumerated using species-specific primers. Results indicated seropositivity rates of 9.69, 9.95, and 10.20% in study population using RBPT reagents of IDEXX-USA, ID.Vet-France, and VRI-Pakistan, respectively, with a statistically nonsignificant difference (p > 0.05). Results of ELISA showed an overall seroprevalence rate of 7.14% in target population with a slightly higher rate in sheep (7.65%) as compared to goat (6.63%) population (p = 0.695; OR = 1.16, 95% CI = 0.53, 2.57). Results revealed that out of total positive samples, B. abortus was detected in 60.71% of seropositive samples and B. melitensis was detected in 14.28% of positive samples. It was revealed that risk factors including body condition scores, hygienic conditions of the housing facility, farming system, reproductive disorders, educational status of farmers, and awareness of farmers about brucellosis had significant association with brucellosis in small ruminants of study area (p < 0.05). Conversely, farm/herd size, locality, gender, age, weight, and parity showed a nonsignificant association (p > 0.05) with brucellosis. In conclusion, brucellosis is prevalent in small ruminants of Multan, Pakistan. It is recommended to devise and implement effective control strategies with a major focus on raising awareness about brucellosis in farmers for the containment of infection in the region.

Applications and Potentials of a Silk Fibroin Nanoparticle Delivery System in Animal Husbandry.

Silk fibroin (SF), a unique natural polymeric fibrous protein extracted from Bombyx mori cocoons, accounts for approximately 75% of the total mass of silk. It has great application prospects due to its outstanding biocompatibility, biodegradability, low immunogenicity, and mechanical stability. Additionally, it is non-toxic and environmentally friendly. Nanoparticle delivery systems constructed with SF can improve the bioavailability of the carriers, increase the loading rates, control the release behavior of the deliverables, and enhance their action efficiencies. Animal husbandry is an integral part of agriculture and plays a vital role in the development of the rural economy. However, the pillar industry experiences a lot of difficulties, like drug abuse while treating major animal diseases, and serious environmental pollution, restricting sustainable development. Interestingly, the limited use cases of silk fibroin nanoparticle (SF NP) delivery systems in animal husbandry, such as veterinary vaccines and feed additives, have shown great promise. This paper first reviews the SF NP delivery system with regard to its advantages, disadvantages, and applications. Moreover, we describe the application status and developmental prospects of SF NP delivery systems to provide theoretical references for further development in livestock production and promote the high-quality and healthy development of animal husbandry.

Insights into midgut cell types and their crucial role in antiviral immunity in the lepidopteran model Bombyx mori.

The midgut, a vital component of the digestive system in arthropods, serves as an interface between ingested food and the insect's physiology, playing a pivotal role in nutrient absorption and immune defense mechanisms. Distinct cell types, including columnar, enteroendocrine, goblet and regenerative cells, comprise the midgut in insects and contribute to its robust immune response. Enterocytes/columnar cells, the primary absorptive cells, facilitate the immune response through enzyme secretions, while regenerative cells play a crucial role in maintaining midgut integrity by continuously replenishing damaged cells and maintaining the continuity of the immune defense. The peritrophic membrane is vital to the insect's innate immunity, shielding the midgut from pathogens and abrasive food particles. Midgut juice, a mixture of digestive enzymes and antimicrobial factors, further contributes to the insect's immune defense, helping the insect to combat invading pathogens and regulate the midgut microbial community. The cutting-edge single-cell transcriptomics also unveiled previously unrecognized subpopulations within the insect midgut cells and elucidated the striking similarities between the gastrointestinal tracts of insects and higher mammals. Understanding the intricate interplay between midgut cell types provides valuable insights into insect immunity. This review provides a solid foundation for unraveling the complex roles of the midgut, not only in digestion but also in immunity. Moreover, this review will discuss the novel immune strategies led by the midgut employed by insects to combat invading pathogens, ultimately contributing to the broader understanding of insect physiology and defense mechanisms.

A study on the epidemiology of brucellosis in bovine population of peri-urban and rural areas of district Multan, southern Punjab, Pakistan.

BACKGROUND: Brucellosis is a zoonotic disease caused by a bacterial pathogen belonging to the genus Brucella. It is one of the most frequent bacterial zoonoses globally but unfortunately, it is still considered as a neglected disease in the developing world. Keeping in view, this study was conducted to determine the prevalence and risk determinants of brucellosis in large ruminants of peri-urban and rural areas of district Multan-Pakistan. For this purpose, blood samples (n = 490) were collected from the cattle (n = 245) and buffalo (n = 245) population of the study area and subjected to preliminary screening of brucellosis using local and imported RBPT reagents. All the samples were further analyzed using commercially available multi-specie indirect ELISA kit followed by their confirmation by PCR using genus and species-specific primers. Data obtained from lab analysis and questionnaires were subjected to statistical analysis for Pearson Chi-square, Odds Ratio and Confidence intervals (95%). RESULTS: The results showed that the maximum seropositivity was recorded with local RBPT reagent (VRI, Pakistan; 12.45%; 95%CI = 9.72-15.65%) followed by RBPT-IDEXX (12.24%; 95%CI = 9.52-15.45%) and RBPT-ID.vet (11.84%; 95%CI = 9.18-14.95%) however statistical difference was non-significant (P = 0.956). The ELISA results showed an overall seroprevalence rate of 11.22% (95%CI = 8.59-14.33%) with comparatively higher rate in cattle (12.65%; 95%CI = 8.82-17.44%) as compared to buffaloes (9.80%; 95%CI = 6.49-14.15%). The PCR analysis confirmed the presence of genus Brucella in all seropositive samples whereas frequency of B. abortus and B. melitensis in seropositive samples was 80% and 20%, respectively. The co-existence of both species was also observed in 5.45% samples. The statistical analysis showed a significant association of bovine brucellosis with herd size, breed, reproductive disorders, mode of insemination, educational status and farmers' awareness about brucellosis (P < 0.05). Conversely, locality, age, weight, gender, pregnancy status, parity and puberty status had no associations with brucellosis (P > 0.05). CONCLUSION: In conclusion, brucellosis is prevalent in large ruminants of district Multan, Pakistan. It is suggested to devise and implement stringent policies for the effective control and prevention of brucellosis in the region. Further, the current situation also warrants the need to strengthen interdisciplinary coordination among veterinarians and physicians in one health perspective to ensure and strengthen the human and animal health care systems in the region.

Prevalence of antibodies to Toxocara canis and its associated risk factors in socio-economically deprived nomadic communities of Pakistan.

Toxocariasis is an important zoonotic disease caused by Toxocara (T.) canis with considerably higher prevalence in developing countries. The data on its epidemiology, especially in socioeconomically deprived nomadic communities, are scarce in Pakistan. Therefore, this study was conducted to determine the prevalence of anti-T. canis antibodies and its associated risk factors in nomadic communities located in and around Multan, Pakistan. A total of 184 sera samples were collected from nomadic communities by simple random sampling technique. The descriptive epidemiological data of participants were collected on well-designed questionnaires. Prior consent was also obtained from the participants to use the data generated from their samples without showing their identity. All the samples were analysed for the detection of anti-T. canis antibodies using commercially available Enzyme-Linked-Immunosorbent-Assay (ELISA) kits having 91% sensitivity and 96% specificity (Bordier Affinity Products, Switzerland). The overall seroprevalence of toxocariasis among nomadic communities was 27.7% (51/184). Various factors, including age, known disease history, nutritional status, contact with dogs, practice of hand washing after contact with dogs, use of unwashed vegetables, body mass index, and drug abuse, showed significant correlation (p < 0.05) with toxocariasis in nomadic communities. Conversely, other factors, including gender, marital status, educational status, awareness about zoonotic diseases, source of drinking water, occupation, location, hand washing before taking food, exposure to soil, and hygienic eating behaviour, showed non-significant correlation (p > 0.05) with seroprevalence of toxocariasis. Results also showed that >50% of seropositive cases were asymptomatic, whereas cough and abdominal pain were recorded in 19.6% and 11.76% of seropositive cases, respectively. Keeping in view, it is suggested to conduct surveys at mass level to rule out the exact disease status at national level and to include nomadic communities in local, national, and regional disease control programs through provision of better healthcare facilities and awareness about the disease.

Bombyx mori nucleopolyhedrovirus induces BmFABP1 downregulation to promote viral proliferation.

Fatty acid binding proteins (FABPs) play an important role as endogenous cytoprotectants. However, studies on FABPs in invertebrates are scarce. Previously, we discovered Bombyx mori fatty acid binding protein 1 (BmFABP1) through co-immunoprecipitation. Here, we cloned and identified BmFABP1 from BmN cells. The results of immunofluorescence indicated that BmFABP1 was localized in the cytoplasm. The tissue expression profile of silkworms showed that BmFABP1 was expressed in all tissues except hemocytes. The expression level of BmFABP1 gradually decreases in BmN cells and B. mori larvae after infection with B. mori nucleopolyhedrovirus (BmNPV). Upregulation of BmFABP1 expression through overexpression or WY14643 treatment significantly inhibited the replication of BmNPV, while downregulation of BmFABP1 expression by RNA interference promoted the replication of BmNPV. The same results were obtained in experiments on silkworm larvae. These results suggest that BmNPV induces BmFABP1 downregulation to promote its proliferation and that BmFABP1 has a potential anti-BmNPV role. This is the first report on the antiviral effect of BmFABP1 in silkworms and provides new insights into the study of the FABP protein family. Also, it is important to study BmNPV resistance in silkworms to breed transgenic silkworms with BmNPV resistance.

Mitigation potential of individual and combined dietary supplementation of local Bentonite Clay and Distillery Sludge against Ochratoxin-A induced toxicity in broilers.

BACKGROUND: This study aimed to evaluate the ameliorative effects of dietary supplementation of local bentonite clay (BN) and distillery sludge (DS) alone and in combination on ochratoxin-A (OTA) induced toxicity in broilers. For this purpose, day-old-broiler chicks (n = 270) were procured from the local market and reared under standard management conditions. After 7 days of acclimatization, birds were divided into 2 main groups A and B with respect to OTA inclusion level in feed, each with four sub-groups viz. A1-A4, each challenged with OTA at a dietary inclusion level of 250 µg/kg feed and B1-B4, each challenged with OTA at the level of 500 µg/kg feed and a common control group that was fed with basal feed throughout the experiment. In groups A and B, BN and DS were administered with feed at the rate of 10 g/kg of feed and 5 g/kg of feed alone and in combination, respectively. RESULTS: Results showed that OTA administration alone resulted in poor feed conversion ratio (FCR) and immunological responses along with increased serum levels of alanine transaminase (ALT), Aspartate transaminase (AST), urea and creatinine (P < 0.05). A significant decrease (P < 0.05) in serum protein levels (albumin, globulin and total protein) was also observed in OTA-fed groups in a dose-dependent manner. The addition of BN at 10 g/kg of OTA-contaminated feed resulted in better FCR and immunological responses as compared to those fed OTA only. The BN supplementation also conferred protection against elevation of serum biochemical parameters when compared with OTA-fed groups. However, the addition of DS could not provide significant protection (P > 0.05) on alteration of serum biochemical parameters in response to the OTA induced toxicity. The combined supplementation of BN and DS resulted in amelioration of OTA-induced toxicity and showed improved FCR, immunological, hematological and serum biochemical parameters (P < 0.05) when compared with other groups. Similarly, BN and DS resulted in a significant decline (P < 0.05) in the OTA tissue residues compared with other groups and control. CONCLUSION: In conclusion, combined dietary supplementation of BN (10 mg/kg) and DS (05 mg/kg) in feed reduced the toxic effects of OTA contamination at levels of 250 and 500 µg/kg of feed in broilers. So, the combination products of BN and DS may be successfully developed for use in poultry for protection against OTA-induced toxicity in broilers.

Seasonal survey, risk factor's analysis and genotyping of Theileria annulata infecting cattle in Punjab province, Pakistan.

Theileriosis is one of the most frequently reported tick borne diseases in tropical and subtropical regions and leads to annual economic losses, such as the reduced dairy products and increased casualties. Tropical theileriosis is caused by Theileria annulata and the present study was designed to improve our knowledge of Theileria annulata infection in Pakistani cattle. In order to assess the prevalence of Theileria annulata on cattle from Multan district in the Punjab province (Pakistan) according to seasons and other risk factors, a total of 1020 blood samples (340 samples each from cross, Holstein Frisian and Sahiwal breed) were collected between 2020 and 2022. Based on Tams-1 partial sequence amplification, the overall T. annulata prevalence was estimated at 11.3% (115/1020). The highest prevalence was observed in autumn season (14.1%), followed by winter (12.9%), summer (11.4%) and spring (6.7%) season. Sahiwal cattle were most susceptible to T. annulata infection (13.2%) followed by Crossbred (11.8%) and Holstein Frisian (8.8%). Epidemiological factor analysis revealed that female cattle, cattle rose with other dairy animals at farm, tick infested cattle, and cattle raised with dogs infested with ticks were associated with the prevalence of T. annulata. White blood cells, lymphocyte (%), Monocyte (%) hemoglobin, mean cell hemoglobin, mean corpuscular hemoglobin concentration, and platelet count were significantly affected blood parameters in T. annulata positive cattle of all three breeds. Representative partial Tams-1 sequences of four Pakistani T. annulata isolates revealed a single genotype genetically close to those infecting cattle from neighboring countries like Iran, Turkey and Egypt. Longitudinal survey and phylogenetic positioning of T. annulata is recommended for epidemiological correlation, diagnosis and treatment of theileriosis in such an agricultural region of Pakistan.

Potential Proteins Interactions with Bombyx mori Nucleopolyhedrovirus Revealed by Co-Immunoprecipitation.

Virus-host interactions are critical for virus replication, virulence, and pathogenicity. The Bombyx mori nucleopolyhedrovirus (BmNPV) is a typical model baculovirus, representing one of the most common and harmful pathogens in sericulture. Herein, we used co-immunoprecipitation to identify candidate proteins with potential interactions with BmNPV. First, a recombinant BV virus particle rBmBV-egfp-p64-3×flag-gp64sp was constructed using a MultiBac baculovirus multigene expression system. Co-immunoprecipitation experiments were then performed with the recombinant BV virus infected with BmN cells and Dazao silkworms. LC-MS/MS analysis revealed a total of 845 and 1368 candidate proteins were obtained from BmN cells and silkworm samples, respectively. Bioinformatics analysis (Gene Ontology, KEGG Pathway) was conducted for selection of proteins with significant enrichment for further confirmation of the effects on BmNPV replication. Overall, the results showed that SEC61 and PIC promoted the replication of BmNPV, while FABP1 inhibited the replication of BmNPV. In summary, this study reveals the potential proteins involved in BmNPV invasion and proliferation in the host and provides a platform for identifying the potential receptor proteins of BmNPV.

Impact of Group II Baculovirus IAPs on Virus-Induced Apoptosis in Insect Cells.

Apoptosis plays an important role in virus-host interactions and is a major element of the insect immune response. Exploring the regulatory mechanisms of virus-induced apoptosis through the expression of apoptotic genes holds important research and application value. Functional research on the reported inhibitor of apoptosis proteins (IAPs) mainly focuses on the group I baculovirus, while the functions of the group II baculovirus IAPs remains unclear. To explore its role in the regulation of the apoptosis of insect cells, we constructed the transient expression vector (pIE1 vectors) and the recombinant baculovirus expressing Bsiap genes (from the Buzura suppressaria nucleopolyhedrovirus) of the group II baculovirus. Apoptosis gene expression results and the virus-induced apoptosis rate show that the overexpression of BsIAP1 could promote apoptosis in insect cells. However, the overexpression of BsIAP2 and BsIAP3 decreases the expression of apoptotic genes, revealing an inhibitory effect. Results on the impact of baculovirus-induced apoptosis also confirm that BsIAP1 reduces viral nucleocapsid expression and the baculovirus titer, while BsIAP2 and BsIAP3 increase them significantly. Furthermore, compared with single expression, the co-expression of BsIAP2 and BsIAP3 significantly reduces the rate of virus-induced apoptosis and improves the expression of nucleocapsids and the titer of offspring virus, indicating the synergistic effect on BsIAP2 and BsIAP3. In addition, combined expression of all three BsIAPs significantly reduced levels of intracellular apoptosis-related genes (including apoptosis and anti-apoptosis genes), as well as apoptosis rate and progeny virus titer, indicating that life activities in insect cells are also inhibited. These findings reveal the relationship between apoptosis and group II baculovirus IAP, which provide an experimental and theoretical basis for further exploration of the molecular mechanism between group II baculoviruses and insect cells.

First report regarding molecular epidemiology and novel variant identification of Anaplasma centrale in cattle from Pakistan.

Anaplasma centrale (A. centrale) is an obligate red blood cell residing tick transmitted rickettsiae that has not been studied extensively for its prevalence in cattle along with its epidemiology. Aim of this investigation was to report the seasonal prevalence, phylogeny and epidemiological parameters associated with the prevalence of A. centrale in cattle breeds enrolled from District Layyah in Punjab, Pakistan. A total of 844 blood samples [Cross breed = 300, Holstein Friesian = 244, Sahiwal breed = 300)] were collected from apparently healthy cattle along with epidemiological data during 2017-18. PCR amplified 426 base pair fragment from 16S rRNA gene of A. centrale in 14.4% (122/844) of cattle. Amplified 16S rRNA partial gene sequence of A. centrale were confirmed by DNA sequencing and deposited to GenBank. Highest A. centrale prevalence was observed in spring (24%) followed by autumn (12.4%) summer (10%) and winter (7.1%) seasons. Sahiwal breed (18.3%) was most susceptible to A. centrale infection followed by cross (12.3%) and Holstein Friesian breed (12.3%). 69/844 (8.2%) of Giemsa stained cattle blood smears were also found positive for Anaplasma spp. Farms where animal use to drink pool water and farms where dogs and other dairy animals were living with cattle had higher A. centrale prevalence. Female cattle and dogs having tick burden were found associated with A. centrale infection. Hematological profile was severely disturbed in A. centrale positive cattle. It is recommended that A. centrale should be screened in cattle, in addition to A. marginale, for the effective control of tick born diseases in Pakistan.

MicroRNA-Mediated Host-Pathogen Interactions Between Bombyx mori and Viruses.

MicroRNAs (miRNAs), small non-coding RNAs of about 22 nucleotides, have been reported to regulate gene expression at the posttranscriptional level and are involved in several biological processes such as immunity, development, metabolism, and host-pathogen interactions. Apart from miRNAs encoded by the host, miRNAs produced by pathogens also regulate host genes to facilitate virus replication and evasion of the host defense responses. In recent years, accumulated studies suggest that viral infections alter the host miRNAs expression profile, and both cellular and viral miRNAs may play vital roles in host-pathogen interactions. Bombyx mori, one of the critical lepidopteran model species, is an economically important insect for silk production. The mechanism of interaction between B. mori and its pathogens and their regulation by miRNAs has been extensively studied. Therefore, in this review, we aim to highlight the recent information and understanding of the virus-encoding miRNAs and their functions in modulating viral and host (B. mori) genes. Additionally, the response of B. mori derived miRNAs to viral infection is also discussed. A detailed critical view about miRNAs' regulatory roles in B. mori-virus interactions will help us understand molecular networks and develop a sustainable antiviral strategy.

Expression and functional characterization in yeast of an endoglucanase from Bacillus sonorensis BD92 and its impact as feed additive in commercial broilers.

Some ingredients used in poultry feed formulation contain carbohydrate polymers which are difficult to digest and thus hinder nutritional feed value. Toward overcoming this limitation, exogenous enzymes have been added to poultry feed to improve its nutritive value. The present study was designed to provide first enzymatic characterization of endoglucanase (BsEgl) from the genome of B. sonorensis BD92 expressed in Pichia pastoris. Further, we tested its impact alone and in combination with a ß-glucosidase (Bteqßgluc) on growth in commercial broilers as feed additive. The expressed enzyme displayed features of GH5 family and had optimum activity against carboxymethyl cellulose at pH 5 and 50 °C. The BsEgl was stable at a range of pH from 4 to 8 for 60 min and at 50 °C for 180 min. Supplementing broilers diet with BsEgl alone or in combination with Bteqßgluc resulted in better feed conversion ratio among treatments during a five weeks testing period. Moreover, meat percentage was also highest for this treatment, and all treatments with recombinant enzymes increased intestinal length in birds compared to treatment control group. Blood parameters and serum biochemistry profile showed non-significant difference among groups. These results support that recombinant cellulolytic enzymes supplement high fiber diets improve their nutritional performance.

Heat Shock Protein 75 (TRAP1) facilitate the proliferation of the Bombyx mori nucleopolyhedrovirus.

The viruses utilize multiple cellular proteins to facilitate their proliferation. The Heat Shock Protein (HSP), the highly conserved protein in eukaryotes and prokaryotes, plays a critical role in facilitating viral proliferation. However, less is known about the role of the HSPs in the life cycles of the Baculoviruses. We constructed recombinant Bombyx mori nucleopolyhedrovirus and discovered the Heat Shock Protein 75 (TRAP1) in the B. mori ovary (BmN) cells by the co-immunoprecipitation experiment using the GP64 (glycoprotein 64) as the bait protein. Tissue expression profile analysis of B. mori indicated that the TRAP1 gene has higher expression levels in the ovary, midgut, and hemolymph. Down-regulation of TRAP1 via RNA interference (RNAi) and geldanamycin (GA, a TRAP1 inhibitor) treatment can reduce the expression level of the major capsid protein VP39 (viral protein 39) of BmNPV. In contrast, the up-regulation of TRAP1 via overexpression can increase the expression level of the VP39. These results indicated that the TRAP1 of B. mori could facilitate the proliferation of the BmNPV. This study provided new insights into the function of TRAP1, and the basic mechanisms of the baculoviruses life cycle for disease prevention.

A Report on Molecular Detection and Phylogenetic Evaluation of Anaplasma marginale in Ticks and Blood Samples Collected from Cattle in District Layyah in Punjab (Pakistan).

Anaplasmosis is a tick-borne disease caused by obligate intercellular gram-negative bacteria, Anaplasma (A.) marginale. The present study reports on seasonal prevalence, epidemiology, and phylogeny of A. marginale in three cattle breeds from District Layyah, Southern Punjab, Pakistan. A total of 844 blood samples (Cross = 300, Holstein Friesian = 244, Sahiwal breed = 300) from apparently healthy cattle on seasonal basis were collected along with epidemiological data during May 2018 till April 2019. Polymerase chain reaction generated 265 base-pair amplicon specific for major surface protein-1b encoding gene of A. marginale in 8.6% (73/844) of enrolled cattle. The highest prevalence was observed during autumn (18.3%) followed by summer (9.7%) and winter season (7.1%). Holstein Friesian breed was most susceptible to A. marginale infection (13.1%) followed by Sahiwal (7.6%) and cross breed (6%). Representative amplified partial gene sequences of A. marginale were submitted to GenBank (Accession numbers MK032842 and MK032843). 37/844 (4.3%) Giemsa-stained blood smears were found positive for Anaplasma spp. Small number of ticks including Hyalomma anatolicum, Hyalomma excavatum, Rhipicephalus microplus, Haemaphysalis punctata were identified from cattle but none of them was found PCR positive for the presence of A. marginale. Analysis of epidemiological factors revealed that female cattle and farm with water supply from pool, farms where other dairy animals and dogs were living with cattle and dogs having ticks load on them had significant association with A. marginale prevalence. It was observed that white blood cell, lymphocytes (%), monocytes (%) hematocrit, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration were significantly disturbed in A. marginale-positive than negative cattle.

Alkaline phosphatase can promote the replication of Bombyx mori cypovirus 1 by interaction with its turret protein.

Bombyx mori cypovirus 1 (BmCPV1) is a member of the Reoviridae family which is characterized by its single-layered capsid. Similar with other turreted viruses in the Reoviridae, transcription of BmCPV1 occurs inside the capsid, and the nascent mRNA is released to the turret which consists of five turret proteins (TPs) and located at the 5-fold axis of the outer capsid, then the capping enzyme TP will guanylate and methylate the nascent viral mRNA to produce a matured mRNA. However, during these processes, how the BmCPV1 draws other cellular proteins to facilitate its replication is still lesser-known. Here we used an ELISA to investigate the interaction between ALP and BmCPV1. A co-immunoprecipitation technique was employed to detect the interaction of ALP with the Methylase domain of TP. We further studied whether ALP affects the replication of BmCPV1 inside the cell, results show that reducing the expression of ALP through RNAi reduced the transcription level of the BmCPV1 VP1 gene, which was increased by overexpression of ALP. In summary, our data demonstrate an interaction between ALP and BmCPV1 and that ALP promoted the replication of BmCPV1, and support our hypothesis of the ALP is an RTPase to facilitate the capping process of BmCPV1.