Induction of cytokines in mice with parainfluenza pneumonia.

The possible involvement of cytokines in the acute viral pneumonia induced by the murine parainfluenza type 1 virus, Sendai virus, was studied. Cytokine profiles for both the respiratory tract and the draining mediastinal lymph node (MLN) of virus-infected C57BL/6J mice were quantified by using the single-cell cytokine (ELISPOT) assay with freshly isolated cell populations and enzyme-linked immunosorbent assay for lung lavage fluids and culture supernatants. Maximal levels of interleukin 2 (IL-2), gamma interferon (IFN-gamma), tumor necrosis factor, IL-6, and IL-10 were detected at the inflammatory site 7 to 10 days after infection, about the time that virus is cleared from the lung. The frequencies of cells producing IL-2, IL-4, IL-6, IL-10, IFN-gamma, and tumor necrosis factor were much higher for the bronchoalveolar lavage (BAL) cell population than for the MLN cell population. Cytokine production after in vitro restimulation of MLN cells was dominated by IL-2 and IFN-gamma, with low levels of IL-10 and IL-6 also being present. Most of the cytokine was produced by the CD4+ cells, although the CD8+ subset was also involved. No IL-4 was found in the BAL fluid or in culture supernatants from restimulated BAL or MLN cells, although a high frequency of IL-4-producing cells was demonstrated in the BAL population by ELISPOT analysis.     

Thermal inactivation of infectious hematopoietic necrosis and infectious pancreatic necrosis viruses.

A plaque assay was used to follow the inactivation kinetics of infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus in cell culture media at various temperatures. Inactivation of infectious hematopoietic necrosis virus in a visceral organ slurry was compared with that in culture media.     

Growth experiment of Hantaan virus in A549 cells: an attempt to improve the immunofluorescent antibody technique for hemorrhagic fever with renal syndrome

An assay method for the infectivity of Hantaan virus, a causative agent of HFRS (hemorrhagic fever with renal syndrome), was developed by the use of IFA (immunofluorescent antibody technique). With the aid of this method, the growth characteristics of Hantaan virus, 76-118 strain, were followed in A549 cells. At a maximal MOI (multiplicity of infection) of 1.6 VAIU (viral antigen-inducing units) per cell, the conventionally available value, plateau level potencies of the viral antigen and virus infectivity were attained at eight and ten days postinfection, respectively, and most of the infective virus produced accumulated in the culture fluids of infected cells. When infections were defined with MOI values in terms of VAIU per cell, development of the viral antigen was highly consistent and followed a given pattern of kinetics. Based on these findings, a protocol for preparation of the viral antigen in IFA was presented, wherein spot culture and FBS treatment were emphasized as effective procedures to minimize non-specific staining.     

Viral adventitious agent detection using laser force cytology: Intrinsic cell property changes with infection and comparison to in vitro testing

Adventitious agent testing in biomanufacturing requires assays of broad detection capability to screen for as many infectious agents as possible. The current gold standard for general infectious adventitious virus screening is the in vitro assay in which test articles are cultured onto a panel of different cell lines and observed for cytopathic effect (CPE). However, this assay is inherently subjective due to the nature of visual observation of cell morphology and labor and time intensive, requiring highly trained personnel to identify CPE. Laser force cytology (LFC) is an alternative, automated analytical method that uses a combination of optical and fluidic forces along with imaging to objectively and quantitatively assess CPE in cell culture. Importantly, because LFC uses no labels or antibodies, the assay is appropriate for general adventitious agent testing. Using LFC, changes in cellular features associated with virally infected cells were identified using principal component analysis. Using these features of infected cells, the sensitivity and earliness of detection with LFC was directly compared with the in vitro assay for a diverse panel of viruses incubated with chinese hamster ovary (CHO), Vero, and Medical Research Council cell strain 5 (MRC-5) cells. LFC detected viral infection with a sensitivity equal to the in vitro assay on average, but in certain virus and cell combinations including mouse minute virus (MMV) and reovirus 3 in CHO cells, detection was 4 days earlier and for MMV, the limit of detection was 10-fold lower. Overall, these results demonstrate the ability of LFC to serve as a biopharmaceutical adventitious agent testing methodology with sensitivity equivalent to the in vitro assay, but in an objective and automated manner.     

Staphylococcal coagglutination, a rapid method of identifying infectious hematopoietic necrosis virus.

A staphylococcal coagglutination test was developed for the rapid detection of infectious hematopoietic necrosis virus (IHNV) in cell cultures and infected fish. The test could be completed in 15 min but required a minimum IHNV titer of 10(6) PFU/ml to obtain a positive reaction. All IHNV isolates, representing the five electropherotypes taken from a wide variety of species and different geographic ranges, caused coagglutination of Staphylococcus aureus cells sensitized with rabbit polyclonal serum against the Round Butte IHNV isolate. The coagglutination reaction was blocked by preincubation of IHNV with homologous antiserum, and IHNV did not cause coagglutination of S. aureus cells sensitized with normal rabbit serum. In specificity tests, cells sensitized with rabbit anti-IHNV serum or normal serum did not coagglutinate in the presence of infectious pancreatic necrosis virus, viral hemorrhagic septicemia virus, cell culture medium components, or media from cultures of cell lines of salmonid and nonsalmonid origin. Most importantly, the coagglutination test was able to detect and identify IHNV directly from experimentally infected rainbow trout fry, the organs of naturally infected adult kokanee salmon and winter steelhead trout, and ovarian fluids of the winter steelhead trout. The coagglutination test is very suitable for field use, since it is inexpensive, simple to interpret, sensitive, and rapid and requires no specialized equipment.     

Staphylococcal coagglutination, a rapid method of identifying infectious hematopoietic necrosis virus.

A staphylococcal coagglutination test was developed for the rapid detection of infectious hematopoietic necrosis virus (IHNV) in cell cultures and infected fish. The test could be completed in 15 min but required a minimum IHNV titer of 10(6) PFU/ml to obtain a positive reaction. All IHNV isolates, representing the five electropherotypes taken from a wide variety of species and different geographic ranges, caused coagglutination of Staphylococcus aureus cells sensitized with rabbit polyclonal serum against the Round Butte IHNV isolate. The coagglutination reaction was blocked by preincubation of IHNV with homologous antiserum, and IHNV did not cause coagglutination of S. aureus cells sensitized with normal rabbit serum. In specificity tests, cells sensitized with rabbit anti-IHNV serum or normal serum did not coagglutinate in the presence of infectious pancreatic necrosis virus, viral hemorrhagic septicemia virus, cell culture medium components, or media from cultures of cell lines of salmonid and nonsalmonid origin. Most importantly, the coagglutination test was able to detect and identify IHNV directly from experimentally infected rainbow trout fry, the organs of naturally infected adult kokanee salmon and winter steelhead trout, and ovarian fluids of the winter steelhead trout. The coagglutination test is very suitable for field use, since it is inexpensive, simple to interpret, sensitive, and rapid and requires no specialized equipment.     

New method for large-scale growth; and concentration of the Epstein-Barr viruses

Efficacious systems are described for the large-scale growth in tissue culture and concentration of infectious (P3HR-1) and transforming (B95-8) Epstein-Barr virus. Also recorded here are our updated procedures for growing stock cultures and protocols to harvest fluids containing biologically active virus which is infectious or transforming. Various methods of concentrating biologically active Epstein-Barr virus have been evaluated. Cellular debris can be removed efficiently and rapidly from culture harvest fluids by clarification through a JCF-Z continuous-flow rotor. Efficient and reliable virus concentration was achieved by molecular filtration with Millipore Pellicon cassettes, using flow rates to 10 liters/h to produce fivefold concentrates followed by pelletization in a fixed-angle rotor. Data from recent production lots showed an average infectivity titer for P3HR-1 virus of 10(4.5) early antigen units per ml (100-fold concentrate) and 10(5.7) transforming units per ml (200-fold concentrate) for B95-9 virus lots.     

New method for large-scale growth; and concentration of the Epstein-Barr viruses.

Efficacious systems are described for the large-scale growth in tissue culture and concentration of infectious (P3HR-1) and transforming (B95-8) Epstein-Barr virus. Also recorded here are our updated procedures for growing stock cultures and protocols to harvest fluids containing biologically active virus which is infectious or transforming. Various methods of concentrating biologically active Epstein-Barr virus have been evaluated. Cellular debris can be removed efficiently and rapidly from culture harvest fluids by clarification through a JCF-Z continuous-flow rotor. Efficient and reliable virus concentration was achieved by molecular filtration with Millipore Pellicon cassettes, using flow rates to 10 liters/h to produce fivefold concentrates followed by pelletization in a fixed-angle rotor. Data from recent production lots showed an average infectivity titer for P3HR-1 virus of 10(4.5) early antigen units per ml (100-fold concentrate) and 10(5.7) transforming units per ml (200-fold concentrate) for B95-9 virus lots.     

马传染性贫血病毒N-连接糖基化回复突变的感染性克隆构建

根据马传贫强毒株EIAV-L和疫苗株EIAV-FDD表面蛋白gp90的N-连接糖基化的变化规律,采用PCR定点突变的方法,对全长感染性克隆pLGFD3-8上的N-连接糖基化的差异区域进行改造后,构建成含有3个N-连接糖基化位点突变的感染性克隆pLGNl91N236N246.将其转染驴胎皮肤细胞(FDD),通过用逆转录酶活性、间接免疫荧光和RT-PCR方法检测而确定其感染性.结果表明,在FDD细胞中盲传三代后,在细胞培养物中可检测到逆转录酶活性,RT-PCR和间接免疫荧光检测均呈阳性,电镜下见到典型的EIAV颗粒.这一结果可能对N-连接糖基化在我国马传贫弱毒疫苗致弱机理的作用研究而奠定良好的基础.     

Application of Equine Infectious Anemia Virus Core Proteins Produced in a Baculovirus Expression System to Serological Diagnosis

Equine infectious anemia virus (EIAV) core proteins were obtained from a baculovirus expression system. Recombinant baculoviruses (rBVs) highly expressed the Gag precursor and p26 antigens in an rBV-infected Sf21 cell culture supernatant. Enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) were conducted using the expressed proteins to detect antibodies from experimentally infected horses. The expressed antigens showed low background levels, high specificity and sensitivity in ELISA and AGID. The results of the serological tests using the expressed antigens were identical to those using a manufactured trial antigen. rBVs containing gag and p26 genes were found to express high quality and large quantities of Gag and p26 antigens, respectively. The antigens were quite useful for detecting anti-EIAV antibodies from virus-infected horses.     

Growth characteristics in cell culture and pathogenicity in mice of two terrestrial rabies strains indigenous to Canada

Two strains of street rabies virus from striped skunks (Mephitis mephitis) were used to infect either a murine neuroblastoma (NA 1300) or a baby hamster kidney (BHK-21/C13) cell culture and the cell infection rates were noted during 4 days postinfection. These cultures were then passaged for four consecutive passages, and the viruses obtained in the supernatant fluids of passage 4 were then treated as original isolates and used to infect both neuroblastoma and baby hamster kidney cells. The mortality period in Swiss white mice caused by the various virus suspensions was noted. The virus strain from the brain of skunks from Saskatchewan infected neuroblastoma and baby hamster kidney cells equally well, produced similar virus titres in supernatant fluids after four subcultures in both cell types, and appeared to produce similar mortality periods in mice from either the original brain tissue or from cell culture supernatant fluids. On the other hand, the virus from the brains of skunks from Ontario readily infected neuroblastoma but poorly infected baby hamster kidney cell cultures. Passage of this strain through four subcultures in both cell types produced virus titres in the supernatant fluids of equal magnitude. However, reisolation of the virus from the supernatant fluid of passage 4 in neuroblastoma cell cultures showed a similar pattern to that from the original brain, while the virus from baby hamster kidney cell passage supernatant fluid was considerably altered. Although the mortality period in mice was similar with virus from the brain and neuroblastoma cell cultures, this period was shortened when mice were inoculated with baby hamster kidney culture supernatant virus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Generalized Immunoassay for Autographa californica Nuclear Polyhedrosis Virus Infectivity In Vitro

A quantitative in vitro immunoassay for the infectivity of Autographa californica nuclear polyhedrosis virus was developed and performed in six different lepidopteran cell lines. The assay was not dependent upon cytopathic effect or polyhedron production, but rather upon viral antigen production and its recognition in a peroxidase-antiperoxidase staining procedure. The importance of using such an assay for accurately assessing infectivity in cell lines which produce polyhedra inefficiently was demonstrated. Differences among the cell lines in sensitivity to viral infection were clearly shown. Differences in the time required to produce infectious progeny were also noted among cells of the same cell line.     

Separation of a Soluble Antigen and Infectious Particles of Bovine Viral Diarrhea Viruses and their Relationship to Hog Cholera

A soluble antigen present in infectious tissue culture fluids was separated from the infective virus particle by ultracentrifugation of two serologically related strains of bovine viral diarrhea viruses, NADL-MD and Oregon C24V. Neutralizing antibodies against the two viruses were absent in four hog cholera antisera, but present in significant titer in the commercially prepared antiserum. Precipitin tests utilizing the agar double diffusion technique formed a single line of identity between the concentrated soluble antigen of both viruses and NADL-MD and hog cholera antisera. No lines were observed using concentrated virus pellet and noninfected BEK cell antigens or control SPF calf and swine sera.     

Persistent infection of a rat kidney cell line with Rauscher murine leukemia virus

Duc-Nguyen, Huu (National Cancer Institute, Bethesda, Md.), Edith N. Rosenblum, and Robert F. Zeigel. Persistent infection of a rat kidney cell line with Rauscher murine leukemia virus. J. Bacteriol. 92:1133-1140. 1966.-The propagation of a murine leukemia virus (Rauscher) in a kidney cell line, derived from a rat with lymphoid leukemia, was studied. A complement-fixing (CF) antigen reacting with Rauscher immune sera was detected at various passage levels, which correlated with the visualization by use of electron microscopy of viral buds and viral particles in different stages of maturation in all passages. Five-month-old monolayers continued to shed virus and to yield high CF antigen titers. The cell-free supernatant fluid from cultures of the 14th passage was shown to be infectious for a normal rat kidney cell line, as evidenced by the appearance of the CF antigen in this line. Interferon production was not demonstrated in infected cultures. The overall data indicated that rat kidney cells could be used to propagate Rauscher virus in a carrier state.     

Biochemical analysis of retroviral structural proteins to identify and quantify retrovirus expressed by an NS0 murine myeloma cell line

A subclone of the NS0 murine myeloma cell line, frequently used to produce recombinant monoclonal antibodies, was found by a transmission electron microscopy method to express a surprisingly high titer of 10(11) retroviral particles per ml of culture supernatant. Infectivity assays showed a very low infectious titer with the restricted host range expected for a murine amphotropic retrovirus. A Western blot assay for the viral capsid protein was developed to confirm the high titer values and provide a means for monitoring batch consistency and virus removal during the purification process. Mass spectrometry of several of the viral Gag proteins demonstrated that the cell line appeared to produce at least two closely related retroviruses. N-terminal sequencing of three of the Gag proteins demonstrated that these retroviruses were members of the murine leukemia retroviral family. Western blot detection with an antibody for the capsid protein gave a linear standard curve over the range of 0.1-3 ng per lane. This allows the detection of viral titers as low as 6x10(7) virions per ml without the need to concentrate the sample. The Western blot method has higher throughput and less variability than transmission electron microscopy methods and has potential for monitoring viral titer and clearance during development of manufacturing processes.     

Rapid detection of SVC virus antigen in infected cell cultures and clinically diseased carp by the enzyme-linked immunosorbent assay (ELISA)

An ELISA was developed using a rabbit antiserum with high levels of binding antibody against SVC virus (SVCV). Modifications were made to the standard assay which increased the sensitivity and resulted in a rapid ELISA (rELISA) which could détéct SVCV antigen in cell cultures and in extracts from infected carp in approximately 1 h. When other cell-grown fish rhabdoviruses were tested, pike fry rhabdovirus (PFRV) antigen cross-reacted in the rELISA but only at a low level. Virus antigen was détécted by the rELISA in clinically and sub-clinically diseased carp during an SVC epizootic but the sensitivity of détéction of subclinical levels of virus was not as great as that of isolation of infectious virus in cell culture. However, antigen was détécted in some fish extracts which failed to yield infectious virus. Fresh extracts of organs from healthy carp were found to give false positive reactions in the rELISA. Kidney and liver extracts from these fish were found to contain a heat labile, non-specific binding factor and further modification of the rapid assay was undertaken which successfully reduced the effect of this factor.