Serum neutralization of nucleopolyhedrosis viruses (Baculovirus subgroup A) pathogenic for Orgyia pseudotsugata

The preparation of antisera to intracellular nonoccluded virions and an in vivo neutralization test procedure (constant serum and virus in dilutions) are described. Results of homologous neutralization tests showed that rabbit antisera to two multicapsid viruses pathogenic for Orgyia pseudotsugata had higher neutralization indices than antiserum to a unicapsid Baculovrus from O. pseudotsugata. Based on reciprocal tests, the three viruses are antigenically distinguishable. Blood serum of rats which had been exposed by inhalation to 25 projected acre doses of a technical-grade Baculovirus preparation demonstrated no viral neutralizing activity. Since the neutralization test used in this study does not require availability of susceptible cell lines and is sensitive and accurate, it could find application in quality control programs and in field monitoring of Baculovirus strains.     

First isolation of an aquatic birnavirus from farmed and wild fish species in Australia

During routine sampling and testing, as part of a systematic surveillance program (the Tasmanian Salmonid Health Surveillance Program), an aquatic birnavirus was isolated from 'pin-head' (fish exhibiting deficient acclimatisation on transfer to saltwater) Atlantic salmon Salmo salar, approximately 18 mo old, farmed in net-pens located in Macquarie Harbour on the west coast of Tasmania, Australia. The isolate grows readily in a range of fish cell lines including CHSE-214, RTG-2 and BF-2 and is neutralised by a pan-specific rabbit antiserum raised against infectious pancreatic necrosis virus (IPNV) Ab strain and by a commercial pan-specific IPNV-neutralising monoclonal antibody. Presence of the virus was not associated with gross clinical signs. Histopathological examination revealed a range of lesions particularly in pancreatic tissue. The virus was localised in pancreas sections by immunoperoxidase staining using the polyclonal antiserum and by electron microscopy. Examination by electron microscopy demonstrated that the virus isolated in cell culture (1) belongs to the family Birnaviridae, genus Aquabirnaviridae; (2) was ultrastructurally and antigenically similar to virus identified in the index fish; (3) is related to IPNV. Western blot analysis using the polyclonal rabbit antiserum confirmed the cross-reactions between various aquatic birnavirus isolates. In addition, PCR analysis of isolated viral nucleic acid from the index case indicated that the virus is more closely related to IPNV fr21 and N1 isolates than to other birnavirus isolates available for comparison. Sampling of other fish species within Macquarie Harbour has demonstrated that the virus is present in several other species of fish including farmed rainbow trout Oncorhynchus mykiss, wild flounder Rhombosolea tapirina, cod Pseudophycis sp., spiked dogfish Squalus megalops and ling Genypterus blacodes.     

Characterization of a virus associated with epidemic conjunctivitis in Thailand.

A strain of virus, named the A-E strain, was isolated from a patient suffering from acute haemorrhagic conjunctivitis during an epidemic of the disease in Bangkok in 1972. The virus had the characteristics of an enterovirus but was not neutralized by any known enterovirus antiserum. Cross neutralization tests indicated that the isolate was closely related to the J670-71 virus isolated in Japan. The virus produced conjunctivitis in rabbits and monkeys following conjunctival inoculation.     

Serological and immunoelectrophoretic relationships among viruses in the tombusvirus group

Serological cross-reactions among eighteen virus isolates of the tombusvirus group were compared in precipitin tube and immunodiffusion serological tests. The isolates were also compared by immunoelectrophoresis in agar gel. Although precipitin tube tests showed considerable and reproducible differences between the various isolates, the results were too greatly affected by other factors to be of value in assessing strain relationships. When pairs of isolates were compared for spur formation in gel-diffusion tests, the results suggested that most isolates could be placed in one of two groups; one group comprised isolates from pelargonium (leaf curl), the other consisted of petunia asteroid mosaic virus and artichoke mottled crinkle virus isolates from Italy and tomato bushy stunt isolates from soil around this Institute and from cherry. Four isolates did not fall into either of these groups; they nearly always formed spurs when compared among themselves, or with viruses in either of the two groups. Pairs of isolates that could be distinguished from each other in spur-formation tests using antiserum homologous to one of them could not always be differentiated when antiserum heterologous to both isolates was used. Immunoelectrophoresis gave consistent results with several methods of virus preparation; it indicated grouping and separation of the isolates in general agreement with the results of gel-diffusion tests: all pelargonium leaf curl isolates were grouped together with slow migration towards the cathode. The petunia asteroid mosaic isolate and the isolates from cherry and from soil from this Institute (GCRI) moved slowly towards the anode. Tomato bushy stunt virus type strain migrated rapidly to the cathode, differing greatly from all other isolates. The method offers a relatively simple means of typing isolates of the tombusvirus group.     

Viral Aggregation Resulting in the Failure to Correctly Identify an Unknown Rhinovirus

A seed lot of strain SF 1684 of rhinovirus type 2 prepared in human embryonic lung cells (WI-38) contained aggregates which interfered with its neutralization by homotypic or homologous antisera. The same virus showed no evidence of aggregation at five other passage levels studied. Virus in the seed lot was not identified correctly, and the titer of homologous antiserum was mistakenly considered to be low as a result of neutralization tests conducted with the aggregated virus. Filtration and a more easily effected treatment with sodium deoxycholate (1%) disaggregated the virus and restored its susceptibility to neutralization by homologous and homotypic antiserum.     

Identification of gp120 regions targeted by a highly potent neutralizing antiserum elicited in a chimpanzee inoculated with a primary human immunodeficiency virus type 1 isolate

We have previously reported that a chimpanzee infected with a primary human immunodeficiency virus type 1 (HIV-1) isolate (HIV-1(DH12)) developed an extremely potent virus-neutralizing antibody. Immunoglobulin G purified from this animal conferred sterilizing immunity following passive transfer to macaques which were subsequently challenged with simian immunodeficiency virus/HIV-1 chimeric virus strain DH12. In addition to being highly strain specific, the chimpanzee antiserum did not bind to the V3 loop peptide of HIV-1(DH12), nor did it block the interaction of gp120 with the CD4 receptor. When neutralization was examined in the context of virus particles carrying chimeric envelope glycoproteins, the presence of all five hypervariable regions (V1 to V5) was required for optimal neutralization. Virions bearing chimeric gp120 containing the V1-V2 and V4 regions of HIV-1(DH12) could also be neutralized, but larger quantities of the chimpanzee antiserum were needed to block infection. These results indicate that the HIV-1 gp120 epitope(s) targeted by the chimpanzee antiserum is highly conformational, involving surface elements contributed by all of the hypervariable domains of the envelope glycoprotein.     

Serological relationship and detection of bean common and bean yellow mosaic viruses in agar gel

Microprecipitin tests demonstrated a distant serological relationship between intact virus particles of BCMV and BYMV. Antiserum titres of 2048–4096 and 16–128 (reciprocals of dilution end-points) were found for homologous and heterologous antigens, respectively. Cross-reactivity, however, was not obtained in agar immunodiffusion tests when the antigens, in purified preparations or crude infective sap, were treated with pyrrolidine or when the reactants were placed in agar gels containing sodium dodecyl sulphate. Only homologous antigens produced a precipitin line; homologous antiserum titre was 16. In comparative immunodiffusion trials, single-radial-diffusion tests (antiserum incorporated in agar) were much more sensitive than double-diffusion tests for detecting low virus concentrations. Analyses of virus proteins by polyacrylamide-gel electrophoresis demonstrated that coat protein of each virus was composed of a single polypeptide chain with a molecular weight of 35000.     

Complement-mediated enhancement of antibody function for neutralization of pseudotype virus containing hepatitis C virus E2 chimeric glycoprotein

We previously reported a number of features of hepatitis C virus (HCV) chimeric glycoproteins related to pseudotype virus entry into mammalian cells. In this study, pseudotype virus was neutralized by HCV E2 glycoprotein-specific antibodies and infected human sera. Neutralization (50% reduction of pseudotype virus plaque formation) was observed with two human immunoglobulin G1 monoclonal antibodies (MAbs) at concentrations of between 2.5 and 10 microg/ml. A hyperimmune rabbit antiserum to an E2 hypervariable region 1 (HVR1) mimotope also exhibited an HCV E2 pseudotype virus neutralization titer of approximately 1/50. An E1 pseudotype virus used as a negative control was not neutralized to a significant level (<1/10) by these MAbs or rabbit antiserum to E2 HVR1. Since HCV probably has a lipid envelope, the role of complement in antibody-mediated virus neutralization was examined. Significant increases in the neutralization titers of the human MAbs (approximately 60- to 160-fold higher) and rabbit antiserum to HVR1 mimotopes (approximately 10-fold higher) were observed upon addition of guinea pig complement. Further, these studies suggested that complement activation occurred primarily by the classical pathway, since a deficiency in the C4 component led to a significant decrease in the level of virus neutralization. This same decrease was not observed with factor B-deficient complement. We also determined that 9 of 56 HCV-infected patient sera (16%) had detectable pseudotype virus neutralization activity at serum dilutions of between 1/20 and 1/50 and that complement addition enhanced the neutralization activity of some of the HCV-infected human sera. Taken together, these results suggest that during infection, HCV E2 glycoprotein induces a weak neutralizing antibody response, that those antibodies can be measured in vitro by the surrogate pseudotype virus plaque reduction assay, and that neutralization function can be augmented by complement.     

The clinico-laboratory characteristics of cases of diseases connected with viruses of the California encephalitis complex in the inhabitants of Moscow

To study the role of viruses of the California encephalitis virus complex (the family Bunyaviridae) in infectious pathology, 187 fever patients admitted to the Clinical Infectious Hospital in May-September 1986 were examined. In 10 of these patients the neutralization test revealed the presence of diagnostically significant changes in neutralizing antibodies (neutralization indices), which was indicative of the role played by Tahyna virus or other related viruses belonging to the California encephalitis virus complex in the etiology of the diseases. The analysis of the clinical picture showed that in all patients the disease took an acute course in its initial stage, starting with shivering and characterized by high fever, headache, pronounced toxicosis, the possibility of the formation of intracerebral hypertension and pneumonia.     

Distribution of marine birnavirus in cultured olive flounder Paralichthys olivaceus in Korea

Surveys of marine birnavirus (MABV) were undertaken in cultured olive flounder Paralichthys olivaceus from the south and west coastal areas and Jeju in Korea during the period January 1999 to April 2007. MABV was detected in all seasons from the fry, juveniles and adult fish from the areas examined. Evident cytopathic effects of the virus including rounding and cell lysis were observed in chinook salmon embryo (CHSE-214) and rainbow trout gonad (RTG-2) cells, but not in fathead minnow (FHM) and epithelial papilloma of carp (EPC) cells. Nucleotide sequences of the VP2/NS junction region of the Korean isolates showed 97.8% ~ 100% similarity, and they belonged to the same genogroup. Cross neutralization tests with serotype-specific rabbit antisera against MABV strains exhibited a close antigenic relationships between strains, and were distinct from infectious pancreatic necrosis virus (IPNV) strains. Coinfection of MABV with bacteria (Streptococcus iniae, Vibrio spp.) and viruses (nervous necrosis virus, lymphocystis disease virus, viral hemorrhagic septicemia virus) was observed.     

Monoclonal antibodies against different epitopes of a 40 Kd capsid protein of infectious bursal disease viruses.

Nine monoclonal antibodies (Mab) against a 40 Kd capsid protein of infectious bursal disease virus (IBDV) strain P3009 were isolated. They were characterized by enzyme-linked immunosorbent assay, indirect fluorescent antibody staining and virus neutralization. They were divided into two groups concerning virus neutralization. Group I Mabs were able to neutralize virus infectivity; however, group II Mabs were not. Competitive binding assays using these Mabs demonstrated the existence of two distinct antigenic regions (A and B) on the 40 Kd protein. Region A was recognized by group I Mabs and region B was by group II Mabs. The binding reaction with group I Mabs was affected by denaturing of the viral proteins, indicating that the antigenic region involving neutralization was conformation-dependent. The results of enzyme-linked immunosorbent assays and virus neutralization tests suggested that group I Mabs might react with one epitope within region A and group II Mabs with 2 or 3 epitopes within region B.     

Immunodiffusion test in avian encephalomyelitis. II. Detection of precipitating antibody in infected chickens in comparison with neutralizing antibody.

The sensitivities of double-immunodiffusion (DID) and neutralization tests to detect avian encephalomyelitis (AE) antibody in chickens were studied. Two antigens were employed in the tests. Concentrated antigen gave a higher titer of antiserum than crude antigen, which reacted only to serum having a neutralization log-index (NI) of 3.4approximately4.0 or more. Antibody responses were examined in four growing chick groups inoculated with AE virus by the intracerebral, subcutaneous and oral routes by the DID test with concentrated antigen and by the neutralization test for 1 or over 2 years after inoculation. When concentrated antigen was used, most sera having an NI of over 1.0 were positive for precipitating antibody. Therefore, the sensitivity of the DID test was nearly equal to that of the neutralization test. The DID test was considered to be applicable to the diagnosis of AE and an antibody survey in the field.     

Cryptic nature of envelope V3 region epitopes protects primary monocytotropic human immunodeficiency virus type 1 from antibody neutralization.

Characterization of biological and immunological properties of human immunodeficiency virus type 1 (HIV-1) is critical to developing effective therapies and vaccines for AIDS. With the use of a novel CD4+ T-cell line (PM-1) permissive to infection by both monocytotropic (MT) and T-cell-tropic virus types, we present a comparative analysis of the immunological properties of a prototypic primary MT isolate of HIV-1 strain JR-CSF (MT-CSF) with those of a T-cell-tropic variant (T-CSF) of the same virus, which emerged spontaneously in vitro. The parental MT-CSF infected only PM-1 cells and was markedly resistant to neutralization by sera from HIV-1-infected individuals, rabbit antiserum to recombinant MT-CSF gp120, and anti-V3 monoclonal antibodies. The T-CSF variant infected a variety of CD4+ T-cell lines, contained positively charged amino acid substitutions in the gp120 V3 region, and was highly sensitive to antibody neutralization. Neutralization and antibody staining of T-CSF-expressing cells were significantly inhibited by HIV-1 V3 peptides; in contrast, the MT strain showed only weak V3-specific binding of polyclonal and monoclonal antibodies. Exposure of PM-1 cells to a mixture of both viruses in the presence of human anti-HIV-1 neutralizing antiserum resulted in infection with only MT-CSF. These results demonstrate that although the V3 region of MT viruses is immunogenic, the target epitopes in the V3 principal neutralizing domain on the membrane form of the MT envelope appear to be cryptic or hidden from blocking antibodies.     

Reduction in plaque size and reduction in plaque number as differing indices of influenza virus-antibody reactions

Jahiel, R. I. (Cornell University Medical College, New York, N.Y.), and E. D. Kilbourne. Reduction in plaque size and reduction in plaque number as differing indices of influenza virus-antibody reactions. J. Bacteriol. 92:1521-1534. 1966.-The serological reactivity of an antigenically hybrid influenza virus recombinant (X-7) was studied in a heteroploid cell plaquing system in which antisera to the parental viruses NWS/(A(0)) and RI/5(+) (A(2)) were incorporated in agar overlay media. Two different effects on plaque formation were found. With NWS antiserum, there was close relationship between reduction in plaque size and in plaque number [plaque inhibition (PI) pattern]. With RI/5 antiserum, plaque size reduction (PSR) occurred over a wide zone of serum dilutions without concomitant change in plaque number (PSR pattern). Several different preinoculation neutralization tests showed a strong reactivity of X-7 with NWS antisera and little if any reactivity with RI/5 antisera. We interpret the differing effects of NWS and RI/5 antisera on X-7 as indicative of the possible occurrence of different mechanisms of neutralization or of the possible participation of different surface antigens. The kinetics of PSR are consistent with the hypothesis that it results from the reaction of RI/5 antiserum with the RI/5-like neuraminidase of X-7. Studies with the antiserum-in-overlay technique of PSR and PI patterns comprise a sensitive method for quantitative antigenic analysis of plaque-forming influenza viruses.     

The Swiss/ICR (Ha) albino mouse as an experimental host for infectious pancreatic necrosis virus of trout

Intracranial inoculation of infectious pancreatic necrosis virus (IPNV), a pathogen of several species of trout, produced pancreatic necrosis in suckling Swiss albino mice. Peri-nuclear halos, cytoplasmic vacuoles, and necrosis were found in histologic sections of pancreas taken from mice killed 21 days post-inoculation. Virus was recovered from the pancreas of mice killed as early as 10 days post-inoculation. Rivers' postulates were fulfilled. Virus recovered from the infected mouse pancreas was neutralized by IPNV specific antiserum. The significance of the mouse as an experimental host is discussed.     

Effects of salmonid fish viruses on Mx gene expression and resistance to single or dual viral infections

We examined the ability of several fish viruses to induce protection against homologous or heterologous viruses in single or double infections, and assessed whether such protection is correlated with innate immunity or expression of the Mx gene. Monolayers of BF2 cells pre-treated with supernatants of brown trout (Salmo trutta L.) macrophage cultures that had been stimulated with either polyinosinic polycytidylic acid (poly I:C) or viruses, such as infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV) or a mixture of the two, showed varying degrees of protection against viral infections. The virus showing the strongest induction was IPNV, and the antiviral activity against IHNV was also high: around 6 log(10) reduction of virus yield. Consequently, the IPNV-IHNV co-infection yield was also reduced by varying amounts. In vivo, the cumulative mortality observed in the IPNV-IHNV co-infected fish was always less than that in those with a single infection. Stimulation with poly I:C for 7 days significantly reduced cumulative mortality in single-infected fish, but not in the double-infected, in which the IPNV was the only virus isolated from moribund animals. By RT-PCR, Mx was expressed in all the organ samples tested (kidney, liver and spleen) from virus-stimulated fish at 1, 2 and 3 days. By qRT-PCR the extent and timing of Mx expression was shown to differ in the poly I:C and the single or dual viral infections. The highest increase in Mx expression (21.6-fold above basal levels) occurred (after 24 h) in fish infected with the IHNV, and expression remained high until day 7. Mx expression in fish infected with IPNV peaked later, at 2 days post infection, and also remained high until day 7. The dual infection with IPNV-IHNV induced high Mx expression on day 1, which peaked on day 2 and remained high until day 7. These results indicate that activation of the immune system could explain the interference and loss of IHNV in the IPNV-IHNV co-infections.     

Hepatitis in skunks caused by the virus of infectious canine hepatitis.

Two cases of acute, fatal, hepatitis occurred in young, striped skunks (Mephitis mephitis) trapped in southern Ontario. Histologically, lesions in the liver were similar to infectious canine hepatitis. A virus was isolated which produced large intranuclear inclusions in dog kidney cell cultures. These inclusions were Feulgen-positive and fluoresced green with acridine orange stain. The skunk hepatitis isolate was identified as the virus of infectious canine hepatitis by virus neutralization tests.     

Localization of a neutralization site of Theiler''s murine encephalomyelitis viruses.

Theiler's murine encephalomyelitis viruses (TMEV) are picornaviruses that produce enteric and neurological diseases in mice. Subgroup TO strains of TMEV cause persistent infections with demyelination, while subgroup GDVII strains neither persist nor demyelinate. We produced neutralizing monoclonal antibodies (mAbs) to clarify the mechanisms of persistence and demyelination. Some of the neutralizing mAbs reacted with isolated VP1 on Western blots, while others were conformation specific. The neutralization site for the former TMEV mAbs was on the VP1 trypsin cleavage site of the intact virion. The neutralization site for the conformation-specific mAbs was distinct and was not affected by trypsin. Trypsin treatment of subgroup TO strains increased their infectivity for L cells, whereas the infectivity of subgroup GDVII strains was decreased by trypsin treatment. Subpopulations of virus in subgroup TO-infected tissue culture cells and in infected mouse brain homogenates contained VP1-cleaved virus; this VP1-cleaved virus gave rise to a large persistent fraction in neutralization tests when it was reacted with VP1-specific mAbs. These findings have implications regarding the pathogenesis of subgroup TO demyelinating disease. TMEV VP1 cleavage may be important for virus persistence because of disruption of a major neutralization epitope. The change in virus surface structure caused by VP1 cleavage may affect cell binding and lead to altered cytotropism. Immunocytes, which have been implicated in subgroup TO demyelination, may provide a source for proteases for VP1 cleavage.