Expression and immunogenicity of glycoprotein H of pseudorabies virus in mammalian cells / 中国生物制品学杂志

@#Objective To express glycoprotein H(gH)of pseudorabies virus(PRV)in mammalian cells and detect its immunogenicity.Methods The gH gene fragment of PRV-XiangA strain was amplified by PCR and inserted into mammalian cell expression vector pIRES-neo3 to construct recombinant expression plasmid pIRES-gH,which was transfected to HEK-293F cells and cultured in suspension for 5 d. The cell culture supernatant was identified by Western blot and purified by nickel ion chromatography column. The purified gH was emulsified with ISA 201 VG adjuvant to immunize 8 female ICR mice,and 8 mice in control group were immunized with the same amount of adjuvant,which was strengthened at 5 and 8weeks after the first dose respectively. The blood samples were collected at 4,7 and 10 weeks after the first dose and detected for the titer of specific antibody and neutralizing antibody in serum of mice;The mice were challenged with PRVXiangA strain(1. 5 × 104TCID50)by nasal drops 2 d after the third blood collection,and observed for the morbidity and mortality daily.Results The recombinant expression plasmid pIRES-gH was constructed correctly as identified by sequencing. The gH protein was successfully expressed and modified by glycosylation in mammalian cells with good reactivity,and about 625 μg purified protein was obtained under 100 mL culture volume. After three times of immunization,mice produced high level of specific antibody and showed the effect of neutralizing PRV,and the titer of neutralizing antibody reached 1∶256. In the challenge test,all the mice in control group became ill and died,while half of the mice in gH immunized group did not get sick with a survival rate of 50%.Conclusion PRV gH was successfully expressed in mammalian cells,and its immune protection was confirmed for the first time,which provided experimental basis for the further research and application of gH,and also provided a new idea for the development of PRV subunit vaccine.

Clinical Spectrum of Primary Polycythaemia and Its Complications -Experience from a Single Center in Karnataka.

Awareness and diagnosis of Philadelphia Negative Chronic Myeloproliferative Disorders has now improved and there is a need for more epidemiological data from India. MethodsThis is a retrospective study of patients of polycythaemia conducted at clinical haematology services, BMCRI, Bengaluru from 2010 to 2017. Results88 patients of polycythaemia were retrospectively studied. 84.1% were male and 15.9% were female. Their ages ranged from 19 to 79 years. 75 (85.23%) had Polycythaemia Rubra Vera (PRV). JAK-2 (V617F) mutation was positive in 33.33%. The commonest presentation was with unexplained erythrocytosis in 50 (66.66%), thrombosis in 20 (26.66%) and with bleeding in 2 (2.66%). 22 thrombotic events occurred in 20 PRV patients. Cortical sinus thrombosis was seen in 27.3%, cerebrovascular accidents in 22.8%, portal vein thrombosis in 13.6%, pulmonary embolism in 9.1%, central retinal artery occlusion in 13.6%, myocardial infarction in 4.5% and digital infarction in 9.1% patients. 3 cases of PRV presented with diplopia. No other definitive cause for ocular palsy could be found. The JAK 2 positive group was slightly older than the negative group and had higher frequency of splenomegaly (p<0.05) and higher values for haemoglobin (p<0.001) and neutrophil counts (p<0.001) and platelet counts (p<0.05). ConclusionsPatients with thrombosis, erythrocytosis, thrombocytosis and haemorrhage should be suspected to have myeloproliferative disorders like PRV and investigated. Ophthalmoplegia is a rare presentation and should raise the suspicion for polycythaemia. There is a higher probability of splenomegaly and higher values for haemoglobin and neutrophil counts and platelet counts in JAK 2 positive group.

Functional analysis of prv-miR-LLT11a encoded by pseudorabies virus

Viral-encoded microRNAs (miRNAs) have vital roles in the regulation of virus replications and host immune responses. The results of previous studies have indicated that miRNA clusters are involved in the replication and virulence of the pseudorabies virus (PRV), which may potentially lead to immune escape or facilitation of PRV replication. This study's previous research revealed that prv-miR-LLT11a was differentially expressed during PRV infection. The present study's results have demonstrated that prv-miR-LLT11a could significantly inhibit PRV replication. It was further determined that SLA-1 was the target gene of prv-miR-LLT11a, and simultaneously, that overexpression of prv-miR-LLT11a could downregulate the mRNA and protein levels of SLA-1 in a dose-independent manner. Furthermore, the present study also observed that prv-miR-LLT11a can downregulate TAP1 expression. Our findings provide a better understanding of the molecular mechanism involved in the effects of prv-miR-LLT11a on SLA-1 and TAP1 as well as its involvement in immune system evasion of PRV.

Tn7-mediated Introduction of DNA into Bacmid-cloned Pseudorabies Virus Genome for Rapid Construction of Recombinant Viruses / 中国病毒学

lacZα-mini-attTn7 was inserted into the intergenic region between the gG and gD genes in a PRV bacterial artificial chromosome (BAC) by homologous recombination in E. coli. The resulting recombinant BAC (pBeckerZF1) was confirmed by PCR and sequencing. Green fluorescent protein (GFP) gene was then transposed into pBeckerZF1 by transposon Tn7 to generate pBeckerZF2. Recombinant viruses vBeckerZF1 and vBeckerZF2 were generated by transfection with the corresponding BAC pBeckerZF1 or pBeckerZF2. The titers and cytopathic effect (CPE) observed for by vBeckerZF1 and vBeckerZF2 was comparable to that of the parental virus vBecker3. vBeckerZF2 was serial passaged for five rounds in cell culture, and the mini-Tn7 insertion was stably maintained in viral genome. These results show that recombinant viruses can be rapidly and reliably created by Tn7-mediated transposition. This technology should accelerate greatly the pace at which recombinant PRV can be generated and, thus, facilitate the use of recombinant viruses for detailed mutagenic studies.

Construction of TK Gene-deleted PRV SH StrainContaining a Single LoxP Site / 中国生物工程杂志

Pseudorabies virus (PRV) is a swine herpesvirus of the Alphaherpesvirinae subfamily and a pathogen of swine resulting in devastating disease and economic losses worldwide. Cre/loxP site-specific system has the character of site specific, time specific, tissue specific and high efficiency in recombination, which makes this system universal in vivo and in vitro recombination of bacteria, fungus, plants, insects and mammals. A recombinant PRV which contain a loxP site in TK locus by using Cre/LoxP recombinant system was construsted. A pair of primers were synthesized according to the pEGFP-C1 sequence published on GenBank, and were used to amplify the EGFP gene expression cassette with two loxP sites flanking each side. This target gene was cloned into pSKLR, the resulting transfer vector pSKLR-GFP-loxP was then cotransfected into 293T cells with PRV SH strain genomic DNA. The recombinant virus rPRV1 was selected and purified in TK-143 cells by choosing fluorescent expressing plaques. Cre expression vector pOG231 was cotransfected into 293T cells with rPRV1 genomic DNA. The second recombinant virus rPRV2 was obtained, which contains only one loxP site in TK locus. Sequencing results of rPRV2 TK gene indicated that 34bp loxP site was inserted into rPRV2 genome and there were 270bp deletion in TK gene. PCR amplifying different generations of rPRV2 TK gene showed that the mutant was stable when passages in RK-13 cells. TCID_ 50 assay indicated that rPRV2 grows well on RK-13 cells. The LD_ 50 test results on BALB/C mice suggested that the virulence of rPRV2 was reduced. As a conclusion, the report gene GFP expression cassette was removed successfully from rPRV1 genome and only one LoxP site was leaved in rPRV2 genome by using Cre/LoxP recombinant system.

Localization of Nerves Innervating Sublingual and Submandibular Gland in the CNS Using Cholera Toxin B Subnit and Pseudorabies Virus / 체질인류학회지

In the rat brain stem, neurons innervating the sublingual and submandibular gland were investigated by means of cholera toxin B subunit (CTB) and pseudorabies virus (PRV). Injection of CTB into the sublingual gland and PRV into the submandibular gland, neural tracer labeled neurons showed similar positions in central nervous system with PRV into the sublingual gland and CTB into the submandibular gland. CTB labeled-neurons were observed in superior salivatory nucleus, PRV labeled-neurons in superior salivatory nucleus and reticular nucleus. CTB was more fine tracer than PRV for observation of superior salivatory nucleus. The size of CTB labeled-neurons is larger in submandibular gland than in sublingual gland. The size of PRV labeled-neurons were nearly the same after injection to submandibular or sublingual gland. No neurons were labeled together with CTB and PRV. Neurons innervating sublingual and submandibular gland were localized independently in superior salivatory nucleus. These results provided a neuroanatomical data of the neurons innervating the sublingual and submandibular gland in the superior salivatory nucleus.

Neural Pathway Innervating Epididymis of Rats by Pseudorabies virus (PRV-Ba-Gal) and WGA-HRP / 대한해부학회지

This experimental studies was to investigate the location of PNS and CNS labeled neurons following injection of 2% WGA-HRP and pseudorabies virus (PRV), beta-galactosidase inserted Bartha strain, into the epididymis of rats. After survival times 4~5 days following injection of 2% WGA-HRP and PRV-Ba-Gal, the rats were perfused, and their brain, spinal cord, sympathetic ganglia and spinal ganglia were frozen sectioned (30 mm). These sections were stained by HRP histochemical and beta-galactosidase histochemical staining methods, and observed with light microscope. The results were as follows : 1. The WGA-HRP labeled sympathetic ganglia projecting to the epididymis were observed in pelvic ganglion and L1-6 lumbar sympathetic ganglia. 2. The WGA-HRP labeled spinal ganglia projecting to the epididymis were observed in L1-6 spinal ganglia. 3. The beta-galactosidase labeled neurons projecting to the epididymis were observed in lamina VII of cervical segments. In thoracic segments, beta-galactosidase labeled neurons were observed in dorsomedial part of lamina I, II and III. Dense labeled neurons were observed in intermediolateral n. and dorsal commissural n.. In lumbar segment, labeled neurons were observed in lamina III, IV, V, dorsal commisural n. and superficial dorsal horn. 4. In the medulla oblongata, beta-galactosidase labeled neurons projecting to the epididymis were observed in the trigeminal spinal n., A1 noradrenalin cells/C1 adrenalin cells/caudoventrolateral reticular n., rostroventrolateral reticular n., area postrema, n. tractus solitarius, raphe obscurus n., raphe pallidus n., raphe magnus n., parapyra-midal n., lateral reticular n. and lateral paragigantocellular reticular n.. 5. In the pons, labeled neurons were observed in Kolliker-Fuse n., locus coeruleus, subcoeruleus n. and A5 noradrenalin cells. 6. In midbrain, labeled neurons were observed in periaqueductal gray substance, retrorubral n., substantia nigra and dorsal raphe n.. 7. In the diencephalon, labeled neurons were observed in paraventricular hypothalamic n., lateral hypothalamic nucleus., medial preoptic n. and retrochiasmatic n.. These results suggest that WGA-HRP labeled neurons of the spinal cord projecting to the rat epididymis might be the first-order neurons related to the viscero-somatic sensory and sympathetic postganglionic neurons, and beta-galactosidase labeled neurons of the brain and spinal cord may be the second and third-order neurons response to the movement of vascular smooth muscle in epididymis. These beta-galactosidase labeled neurons may be central autonomic center related to the integration and modulation of reflex control linked to the sensory and motor system monitoring the internal environment. These observations provide evidence for previously unknown projections from epididymis to spinal cord and brain which may be play an important neuroanatomical basic evidence in the regulation of epididymal function.

Tracing Study on Neuroaxis of Medial Rectus Muscle in the Brain Stem of the White Rat

To clarify neuronal connection of the medial rectus muscle at brain stem, Bartha strain of the psecudorabies virus(PRV-Ba) and cholera toxin-horeserdish peroxidase(CT-HRP) was injected into medial rectus muscle of the rat. About 84 hours after PRV or CT-HRP injection, the brain was removed and processed immunohistochemical stain for PRV-Ba and neurohistochemical stain for CT-HRP using tetramethyl benzidine. The CT-HRP positive reaction was only present in ipsilateral oculomotor nucleus, contralateral abducence nucleus and bilaterally in rostral interstitial nucleus of medial longitudinal fasciculus, olivary pretectal nucleus, medial and superior vestibular nucleus, nucleus prepositus hypoglossi, prerubral field, nucleus Darkschewitsch, lateral substantia nigra, terminal nucleus of accessory nucleus, dorsal raphenucleus, locus ceruleus and pontime reticular formation. This study confirmed that both oculomotor nucleus and abducence nucleus were connected with each other through the medial longitudinal fasciculus, and neuronal connection of the medial rectus muscle in the rat brain stem.

Comparison between CT-HRP and Bartha Strain of Pseudorabies Virus as a Neurotracer in the Rat Visual System

To investigate the applicability of the Bartha strain of pseudorabies virus(PRV-Ba) as a neurotracer in the visual system, we performed immunohistochemical study of the rat brain after intravitreal injection of the PRV-Ba. Cholera toxin-horseradish peroxidase(CT-HRP), a well known neurotracer, was also injected into the vitreous cavity for comparison. After given survival time the brain was removed and processed immunohistochemical stain for PRV-Ba and neurohistochemical stain for CT-HRP. The CT-HRP positive reactions were observed almost contralaterally in optic chiasm, optic tract, suprachiasmatic nucleus(SCN), olivary pretectal nucleus(OPT), all three subdivision of lateral geniculate body, superficial gray layer and optic layer of superior colliculus and medial, lateral, dorsal terminal nucleus of accessory optic tract. PRV-Ba immunopositive reaction was observed in SCN and OPT, but not in nerve fiber system and dorsal lateral geniculate body. Also PRV-Ba positive cells were distributed mainly in intermediate layer and weakly distributed in superficial layer of the superior colliculus. This study suggests that PRV-Ba is not a useful neurotracer in studying the visual system of the rat.