Crude Virus Preparations Research Articles

Designing an in‐house ELISA to detect antibody against viral haemorrhagic septicaemia virus using recombinant N protein in Iranian farmed rainbow trout ( Oncorhynchus mykiss )

Viral haemorrhagic septicaemia (VHS) is one of the most important viral diseases in rainbow trout that has caused great losses to Iranian rainbow trout aquaculture industry in the last 3 years. Therefore, rapid and reliable diagnosis of VHS virus infections is of great importance. An enzyme linked immunosorbent assay (ELISA) method was performed to study serum antibodies against viral haemorrhagic septicaemia virus (VHSV) using recombinant fragments of their N protein. For this purpose, the virus was first isolated from an infected farm. A part of the nucleocapsid (1–505 bp) gene was amplified by RT-PCR using specific primers. The amplified fragment was ligated to pMALc2x vector and transferred to DH5α strain of Escherichia coli. Then, recombinant plasmids were tested for protein expression in E. coli Rosetta strain. SDS-PAGE analysis indicated the production of a recombinant protein with an expected molecular weight of 61 KDa. Analysis of trout serum samples from seven previously infected farms and two VHS free farms showed that the designed ELISA method was effective in diagnosing the infected fish. The results revealed that the developed serological assay using designed ELISA based on recombinant protein (N) has the potential to be used in monitoring studies and to determine the prevalence of VHS in rainbow trout farms. The present data allow evaluating the levels of nonneutralizing antibodies without crude virus preparations.

Development and validation of a HPLC method for the quantification of baculovirus particles

A HPLC method using an anion exchange column was developed for the quantification of baculovirus particles. To properly detect the virus eluting from the column, a nucleic acid dye was used to amplify the signal projected by the virus. The viral genome was labeled by incubating the virus with SYBR Green I at 37°C for a minimum of 1h. The virus was specifically eluted from the contaminants in 8.9min at a NaCl concentration of 480mM NaCl (in 20mM Tris–HCl, pH 7.5). The total run time of the method was 25min. The method resulted in a linear response from 1×108 to 5.0×1010viral particles (VP/ml). The detection limit was 3.0×107 and the quantification limit was 1×108VP/ml. The intra-assay precision was <10% for both purified and crude virus preparations whereas the inter-assay precisions were <5% and <10% for purified and crude virus preparations, respectively. The recovery/accuracy of the method ranged from 78 to 101%. This method is a robust monitoring tool to facilitate research activities with baculovirus vector and accelerate development of baculovirus-based processes for manufacturing of biologics.

Bio-suppression of coconut rhinoceros beetle, Oryctes rhinoceros L. (Coleoptera: Scarabaeidae) by Oryctes baculovirus (Kerala isolate) in South Andaman, India

Coconut rhinoceros beetle ( Oryctes rhinoceros L.) is the major constraint in the production of coconut across the Andaman & Nicobar Islands. To suppress the pest population, baculovirus was released for the first time in 1987 at four locations in South Andaman, which brought down palm damage to insignificant levels within 48 months of release. However, during 1999–2000 large-scale felling of old coconut palms to be replaced with fresh ones by the Andaman Plantation Development Corporation (APDC) resulted in a fresh outbreak of coconut rhinoceros beetle. This warranted release of virus through infected beetles to suppress the pest population. A dose of 130 μg crude virus preparation (CVP)/beetle ensured optimum longevity coupled with low fecundity for efficient dispersal of virus. Release of such pre-infected beetles (795) at five locations in South Andaman resulted in over 90% reduction in palm damage within 23 months of release. Examination of experimental and adjoining palm groves confirmed the prevalence of virus among the sampled beetle population, which registered three-fold increases since the release of virus. There was a drastic reduction to the extent of 91% in the occupancy. However, the recent tsunami in December 2004 in addition to destroying 5000 ha of coconut area has generated huge amounts of organic matter, which has resulted in the rhinoceros beetle outbreak. Augmentation of virus is suggested for effective management of the pest.

Acute Effects of Purified and UV-Inactivated Herpes simplex Virus Type 1 on the Hypothalamo-Pituitary-Adrenocortical Axis

Herpes simplex virus type 1 (HSV-1) is a common cause of viral encephalitis, manifested by neuroendocrine and behavioral changes. We have previously demonstrated that HSV-1 induces marked hypothalamo-pituitary-adrenocortical (HPA) axis activation. In this study we characterized the acute effects of HSV-1 on the HPA axis occurring before viral replication and appearance of clinical signs of encephalitis. Since in previous studies we used crude virus preparations which may contain immune factors produced by the infected cells, we tested here the effects of purified HSV-1 virions. HSV-1 was propagated on Vero cells and virions were purified by centrifugation in sucrose gradients. Inactivation of viral infectivity was achieved by UV-irradiation, which caused a million-fold decrease in virus titer, as determined by plaque assay. Intracerebroventricular (ICV) inoculation of crude or purified virions induced a dose dependent increase in serum corticosterone and corticotropin (ACTH). This effect was maximal within 3.5 h postinfection and lasted for 72 h. ICV inoculation of UV-inactivated purified virions caused a marked increase in serum corticosterone and ACTH at 3.5 h, but in contrast to the effect of the active virus, the hormone levels gradually decreased at 24 h, and returned to basal levels at 72 h postinfection. HSV-1-induced HPA axis activation at 3.5 h was completely abolished by pretreatment with interleukin-1 receptor antagonist, injected ICV. Adrenalectomized rats failed to respond to ICV inoculation of purified HSV-1 by increase in ACTH. In contrast, these rats responded to ICV injection of LPS. In conclusion: (1) HSV-1 can acutely activate the HPA axis before and independently of any viral replication; (2) HSV-1-induced HPA axis activation depends on a permissive action of circulating glucocorticoids and on host derived brain interleukin-1.

Western blot (immunoblot) assay of small, round-structured virus associated with an acute gastroenteritis outbreak in Tokyo

Small, round-structured virus (SRSV) was detected in a stool specimen of a patient during an acute gastroenteritis outbreak in Tokyo and was tentatively named SRSV-9. SRSV-9 was purified by sucrose velocity gradient centrifugation after CsCl density gradient centrifugation. The buoyant density of SRSV-9 appeared to be 1.36 g/ml in CsCl. A Western blot (immunoblot) assay using the biotin-avidin system revealed that SRSV-9 was antigenically related to the Hawaii agent but distinct from the Norwalk agent and contained a single major structural protein with a molecular size of 63.0 +/- 0.6 kilodaltons. The prevalence of SRSV-9 infection in Tokyo was surveyed by the Western blot antibody assay by using a crude virus preparation as the antigen. Seroconversion was observed in 56.5% of the patients involved in the outbreaks from which SRSV was detected by electron microscopy.

Time-resolved immunofluorescence: a sensitive and specific assay for anti-HIV antibody detection in human sera

We describe a new immunoassay, time-resolved fluoroimmunoassay (TR-FIA), for detection of anti-HIV antibodies in human sera. This method is based on the use of a crude virus preparation coated on a polystyrene microtitre plate and of a swine anti-human IgG labelled with a rare earth metal, europium, as fluorescent label chelated with EDTA derivatives. A light pulse from a xenon lamp (340 nm) was used to excite the label and after a 400 μs delay time the emission fluorescence was counted for 400 μs at 613 nm. This cycle was repeated 1000 times during the total counting time of 1 s. TR-FIA presents considerable advantages over other techniques: (a) it avoids time-consuming, expensive and hazardous virus purification steps; (b) it excludes the use of radiotracers or substrates with potential health risks to reveal the reaction; (c) it has high sensitivity and specificity. A total of 475 serum specimens were tested by ELISA and by TR-FIA. The proportions of positivity were 29.6% by ELISA versus 26.7% by TR-FIA. The sensitivity of both systems was 100%. The specificity was 87.5% for ELISA, whereas it reached a value of 99.4% for immunofluorimetric assay.

Decline in infectivity of simian virus 40 by sodium deoxycholate and its restoration with the extract of monkey kidney cells

Plaque-forming activity and T-antigen-synthesizing activity in the crude preparation of simian virus 40 (SV40) decreased to 1/20–27 after treatment with 0.5% sodium deoxycholate (DOC) for 30 min at 37°C. A full restoration of the activity occurred after incubation of DOC-treated virions with the extract of monkey CV-1 cells, host cells for productive infection with SV40. Analysis by sedimentation through 15% sucrose to CsCl cushion (ϱ = 1.327 g/cm 3) revealed that virions in the [ 35S]methionine-labeled crude virus preparation sedimented to the interface between CsCl and sucrose, and that treatment with DOC resulted in the loss of infectivity and the appearance of virions sedimentable into CsCl cushion. The [ 35S]methionine-labeled purified virions (prepared after treatment with DOC and sedimentable into CsCl cushion) sedimented to the CsCl-sucrose interface after incubation with the cell extract, with restoration of infectivity. The infectivity-restoring activity of the cell extract was sensitive to ethyl ether, partially sensitive to heating at 75°–97° for 30 min, but resistant to treatment with DNase (50 μg/ml), RNase (40 μg/ml), or trypsin (0.05%) for 30 min at 37°. These results suggest that lipid-related cellular components bind stably to virions of SV40 and facilitate an efficient infection.

Immune complexes in Aleutian disease: Demonstration of antibody on isolated virus

The specific binding of Staphylococcal protein A for mammalian immunoglobulin G was used to demonstrate IgG associated with Aleutian disease virus (ADV) when isolated from infected mink tissues. Protein A specifically bound to mink serum Ig with no reaction with other serum or tissue proteins. Protein A labeled with 131Iodine reacted with crude virus preparations but not with virus that had been purified by freon extraction to the point where it became reactive with antibody by counterimmunoelectrophoresis. Binding to purified ADV was restored when the purified virus was first reacted with antibody. Results of urea treatment indicated this as an alternative method for isolation of ADV free from antibody.

Reverse transcriptase of foamy virus. Purification of the enzymes and immunological identification.

Reverse transcriptase from foamy virus, strain H4188 was estimated and purified. The enzyme has the following characteristics: 1. The reaction utilized preferentially oligo (dT) poly (rA) as a primer-template; however, the synthetic primer-template oligo (dT) poly (dA) could also be used to some extent. 2. The reaction utilized oligo (dG) poly (rC) as a primer-template with very low efficiency. 3. The crude virus preparation had a detectable endogenous reaction using the four deoxyribonucleotides for DNA polymerization. 4. The cation requirement for the enzyme reaction was much more biased for Mn++ than for Mg++ ions. 5. The molecular weight of the partially-purified enzyme was estimated to be about 80,000. Aggregates of 240,000 daltons were also seen. The activity of this enzyme was not inhibited by antisera against the reverse transcriptases of various type C RNA viruses, namely, feline endogenous leukemia virus, RD 114, Woolly simian sarcoma virus (SSV-1) and avian myeloblastosis virus (AMV). Antiserum against Rauscher leukemia virus (RLV) enzyme was marginally active against foamy virus enzyme, perhaps indicating a slight cross-reaction. The biochemical characteristics of foamy virus reverse transcriptase seemed to be very close to those of the type C RNA viruses, but the immunological reaction proved that the foamy virus reverse transcriptase was distinct from the others.

Comparitive study of the milk fat globule membrane and the mouse mammary tumour virus prepared from the milk of an infected strain of Swiss albino mice

Milk fat globule membranes and mammary tumour virus particles ( d = 1.17 g/cm 3 ) have been obtained from the milk of a Swiss albino mice strain. Comparitive biochemistry shows that these two structures differ significantly in the phospholipid, polypeptide and glycopolypeptide patterns and enzymatic activities. However, the lipid profile and the morphology of both structures suggest a filiation with the plasma membrane. Density fractions obtained from the crude virus preparation have been thoroughly investigated. The results suggest that most of these fractions represent degraded virus and/or atypical virus assembly.

Absence of glycoproteins in poliovirus particles.

SUMMARY Properly purified preparations of poliovirus particles are practically free of d-galactose and N-acetyl-d-glucosamine. These sugars, however, are found in crude virus preparations, obtained by high and low speed sedimentation and by CsCl-gradient fractionation from infected HeLa cells. They are separated from the virus particle by extraction with chloroform and subsequent isopycnic sedimentation of the virus preparation. Since d-galactose and N-acetyl-d-glucosamine are regular constituents of glycoproteins, the absence of significant amounts of these sugars demonstrates the lack of glycoproteins in poliovirus particles. Radioactive monosaccharides are recommended for the detection of cellular impurities in carbohydrate-free viruses. A simple and rapid method for the purification of large quantities of poliovirus by ‘precipitation’ with polyethylene glycol, resuspension in CsCl solutions, extraction with chloroform and isopycnic sedimentation is described.

Differentiation of four adenovirus types by macrophage migration inhibition tests.

The macrophage migration inhibition (MMI) test was found to be a satisfactory procedure for distinguishing between adenovirus types 1, 4, 5, and 7. Highly purified virus preparations were used for the sensitization of Hartley strain guinea pigs, whereas the MMI test antigen consisted of crude virus preparations grown in KB cells. With all four virus types, a significantly greater MMI response was noted when peritoneal exudate cells were exposed to the homologous sensitizing antigen as compared to that obtained with the three heterologous antigens. Studies with adenovirus type 1 indicate that sensitizing doses between 70 and 150 mug of viral protein per guinea pig gave the optimal MMI response. Doses below 70 mug did not stimulate the delayed response, whereas doses above 120 mug produced MMI reactions which were nonspecific, as differences between homologous and heterologous antigens were not demonstrable.

The accumulation and elimination of crude and clarified poliovirus suxpensions by shellfish.

Hoff, J. C. and R. C. Becker (Northwestern Water Hygiene Lab., Rt. 4, Box 4129, Gig Harbor, Wash. 98335). The accumulation and elimination of crude and clarified poliovirus suspensions by shellfish. Amer. J. Epid., 1969, 90: 53–61.— The accumulation and elimination of Escherichia coli and poliovirus type 1 (LSc2ab) by the Olympia oyster (Ostrea lurida), Pacific oyster [Crassostrea gigas), and Manila clam (Tapes japonica) were studied in a flowing seawater system. E coli and crude virus preparations containing cell debris were concentrated by all three species to much higher levels than were clarified virus suspensions from which the larger particulate matter had been removed. Virus accumulation ratios (concentration in shellfish/concentration in water) ranged from 10- to 900-fold with the use of crude virus and from 0.4- to 3.6-fold with clarified virus preparations. Both crude and clarified virus preparations were eliminated by the shellfish at similar rates. Virus concentrations declined rapidly during the first 24 hours, then continued to decline more slowly. Clarified virus was usually eliminated to indetectable levels within 48 hours whereas crude virus, originally accumulated to much higher levels, was frequently detectable after 72 or 96 hours. The results indicate that the state in which viruses exist in natural waters, i.e., whether they are present as free particles or attached to larger particulate matter, is an important consideration in assessing the potential of shellfish for the transmission of enteroviruses.

The polypeptides of adenovirus: II. Soluble proteins, cores, top components and the structure of the virion

The adenovirion has been shown to have a much more complex structure than was previously assumed. Eight different types of polypeptides have been assigned a place in the particle. Three are present in an outer capsid, three in an inner core, and two others in association with hexons. The hexon capsomere is composed of about three molecules of a single type of peptide of molecular weight (MW) 120,000 which comprises about 50% of the total virion protein. Groups of such capsomeres, released from the virion by 5 M urea, are found to be associated with two minor polypeptides, each of MW 13,000. The penton-base is composed of a single type of peptide of MW 70,000, and the fiber is composed of another peptide of MW 62,000. The internal core released by treatment of the virion with 5 M urea, contains the viral DNA in association with three arginine-rich peptides of MW 44,000, 24,000, and 24,000, comprising some 20% of the total virion protein. The same three peptides are relatively lacking in the “empty” capsids found as the “top components” of cesium chloride gradients of crude virus preparations.

Inactivation of vaccinia virus by gamma radiation.

The influence of the purification on the inactivation of Vaccinia Virus (VV) by gamma radiation has been studied. Differential centrifugation and sucrose density gradient centrifugation have been used. Purification modifies the VV compound survival curve previously published 1 2. The possibility that multiple reactivation might be the reason of compound survival curve when crude virus preparations were irradiated has also been studied, but the experiments made give negative results. Even with the best method of purification applied, the target volume of about 10-17 cm3 for VV calculated from the irradiation experiments is very small compared to the volume of the virus obtained with the sedimentation methods.

A study of the herpes simplex virus-rabbit kidney cell system by the plaque technique

Herpes simplex virus on rabbit kidney monolayers forms plaques, 1 to 2 mm in diameter, in 4 to 6 days. There is a linear relationship between the virus concentration and the number of plaques. Approximately 50% of the virus is adsorbed on monolayers in 90 minutes. The titer of virus as determined by plaque count is 10 to 100 times higher than the titer on the chorioallantoic membrane of chick embryos. A study of some of the physical properties of herpes virus showed that less than 1% of the virus survives incubation at 37° for 24 hours. The virus is extremely sensitive to ether. It is quite resistant to sonic vibration and to freezing and thawing; these procedures actually increase the titer of crude virus preparations. Growth curves in monolayer cultures reveal a latent period of 8 hours and an average virus yield per cell of 280 plaque-forming units (pfu). When cells infected with virus:cell ratios of 1.6 or 0.22 were studied in dilute suspension, the latent period was 7 hours and there was an increase in the final yield by a factor of 100 over the latent period. When the virus:cell ratio was increased to 14 or 64, the latent period was reduced to 5 hours and the average factor of increase was 125. Sonic vibration of suspended cells infected with virus:cell ratios of 3 to 4 showed that intracellular virus makes its first appearance about 5 hours after infection; the average release time of intracellular virus was calculated to be 2.3 to 3.5 hours. The ability of rabbit kidney cells to support the growth of herpes virus is very resistant to irradiation with X-rays.