Interferon induction and sensitivity as correlates to virulence of Venezuelan encephalitis viruses for hamsters

Venezuelan encephalitis (VEE) virus strains, which differ in virulence for adult hamsters, were compared with respect to (a) sensitivity to hamster interferon (IF) in vitro and in vivo and (b) induction of IF in plasma and target tissues (spleen, bone marrow and brain) following subcutaneous inoculation. In vitro, in cultures of a continuous line of hamster kidney cells, hamster interferon inhibited the replication of a benign VEE strain (BeAr 35,645) more than another benign strain (TC-83 vaccine) or two hamster-virulent strains (68 U 201 and Trinidad donkey). In vivo, in hamsters given poly I: poly C 24 hours before virus to induce interferon formation, BeAr 35,645 and TC-83 virus infections were prevented more frequently than infections with virulent strains Trinidad donkey and 68U201. Benign VEE strains BeAr 35,645 and TC-83 induced only slightly lower concentrations of IF in plasma, bone marrow, spleen and brain than virulent strains Trinidad donkey, 63Z21 and 68U201. However, concentrations of infectious BeAr 35,645 virus were significantly lower in these tissues than virulent strains, resulting in higher ratios of IF: invectious virus, suggesting efficient interferon induction. Benign strain TC-83 showed irregular relationships between IF and infectious virus in plasma or blood and tissues, Splenectomy significantly depressed plasma plasma IF responses to TC-83 virus 20 to 30 hours after inoculation. Interferon appears to be a factor that influences virulence of VEE viruses for hamsters.     

Isolation of a temperature-sensitive dengue-2 virus under conditions suitable for vaccine development

Dengue virus, type 2, in viremic human sera and after passage in cell cultures produces mixtures of small and large plaques when assayed in LLC-MK2 cells. Clones of dengue virus type 2 obtained by plaque selection in primary green monkey kidney cell cultures were tested for temperature sensitivity in vitro and for virulence by intracerebral inoculation of suckling mice. Sublines of a small-plaque clone were found to have lower nonpermissive temperatures than the parent virus by both plaque formation and release of infectious virus into the culture media. Small-plaque sublines were significantly less virulent in suckling mice than was the parent virus. Sublines from a large-plaque clone were not temperature sensitive and closely resembled parent virus mixed-plaque morphology. When small-plaque sublines were serially passaged using undiluted inocula, reversion occurred as evidenced by the appearance of large plaques and return of mouse virulence. Small-plaque virus could be maintained through several serial passages without reversion by using low-input inocula. Desirable passage history as well as temperature-sensitive and attentuation characteristics of the S-1 small-plaque subline make it appear suitable as a vaccine candidate virus.     

Characterization of the epidermal growth factor receptor in pig oviduct and endometrium

Two different plaque variants of Japanese encephalitis virus were selected from a wild-type Taiwanese isolate using Vero cells. One variant was found to exhibit small plaque morphology with retarded virus replication kinetics in Vero cells, and was demonstrated to be resistant to monoclonal antibody (mAb) E3.3 neutralization. The other variant showed large plaque morphology, was sensitive to mAb E3.3 neutralization, and manifested reduced virulence in mice on both intracranial and intraperitoneal inoculations. These two variants propagated in Vero cells retained high levels of infectivity but had relatively low HA titers as compared with the parent strain. The envelope sequences of these two variants showed four amino acid differences at residues E-85 (Glu/Arg), E-306 (Glu/Gly), E-331 (Ser/Arg), and E-387 (Met/Arg). Our results indicated the neutralizing epitope of Japanese encephalitis virus did not overlap with virus virulence determinant.     

Deletions and duplication in internal inverted repeat sequence of long region/unique sequence of long region (IRL/UL) of herpes simplex virus type-1 (HSV-1) genome are not evidently associated with intracranial and foot-pad pathogenicity in mouse model

The biological properties of three deletion variants (1704, 1705 and 1706) of herpes simplex virus type-1 (HSV-1) strain 17 syn+, were studied by establishing a base line pathogenicity of nine individual plaques from the parental 17 syn+ elite stock. Restriction enzyme analysis of deoxyribonucleic acid (DNA) from each of the nine plaque stocks and intracranial inoculation into three weeks old BALB/c mice showed no difference in the size of fragments and distribution of the sites or their 50% lethal dose (LD50) values [plaque forming units (pfu)/mouse] as compared to the parental 17 syn+ stock. Inoculation of the variants into three weeks old BALB/c mice showed that 1705 was not different in pathogenicity from the wild type following intracranial and footpad inoculations. On the other hand variants 1704 and 1706, when compared to the wild type virus were less virulent on intracranial inoculation i.e. the difference in LD50 values was approximately one log and two logs respectively and both the variants failed to kill any of the animals following footpad inoculation even at the dose of 1 x 10(7) pfu/mouse. During in vivo replication experiment in the peripheral nervous system of mice, 1704 and 1706 grew very poorly.     

Virulence, immunogenicity and reactivation of bovine herpesvirus 1 mutants with a deletion in the gC, gG, gI, gE, or in both the gI and gE gene

Within the framework of developing a marker vaccine against bovine herpesvirus 1 (BHV1), several mutants with deletions in non-essential glycoprotein genes were constructed. Glycoprotein gC, gG, gI and gE single deletion mutants, a gI/gE double deletion mutant and a gE frame-shift mutant were made. The virulence and immunogenicity of these mutants were evaluated in specific-pathogen-free calves. Except for the gC deletion mutant, all mutants were significantly less virulent than the parental wild-type (wt) BHV1 strain Lam. The virulence of the gI and the gI-/gE- mutants was almost completely reduced. Upon challenge infection, the calves of the control group became severely ill, whereas all other calves remained healthy. The reduction of the virus shedding after challenge infection was related to the virulence of the strain of primary inoculation. Virus shedding was almost completely reduced in calves first inoculated with Lam-wt or with gC- and the least reduced in calves inoculated with gI- or gI-/gE-. Six weeks after challenge, all calves were treated with dexamethasone to study whether mutant or challenge virus or both could be reactivated. The gC- and the gG- mutants were reactivated, whereas none of the other mutants were reisolated. Reactivation of challenge virus was reduced in all calves inoculated with mutant viruses. The gC deletion mutant was too virulent and the gI and the gI/gE deletion mutants were the least immunogenic, but based on residual virulence and immunogenicity, both the gG and the gE deletion mutants are candidates for incorporation in live BHV1 vaccines. However, it also depends on the kinetics of the anti-gG and anti-gE antibody response after wild-type virus infection, whether these deletion mutants are really suitable to be incorporated in a marker vaccine.     

Characterization of influenza A/HongKong/156/97 (H5N1) virus in a mouse model and protective effect of zanamivir on H5N1 infection in mice

A recent outbreak of influenza in Hong Kong was caused by a highly virulent virus of avian origin. Concern that the appearance of such a virus in the human population may be a harbinger of a new pandemic has brought increased attention to the issue of antivirals available for treatment of influenza. A/HongKong/156/97 (H5N1), the first virus of H5N1 subtype isolated from a human host, is highly virulent in the mouse model and can infect mouse lungs without requiring adaptation. High mortality and evidence of systemic disease, including spread to the brain after intranasal inoculation, are observed. Zanamivir, a novel neuraminidase inhibitor, is effective at decreasing replication of the virus in vitro. In a model of lethal challenge in mice, zanamivir reduces lung titers of the virus and decreases morbidity and mortality.     

Herpes simplex virus type 1 glycoprotein gC mediates immune evasion in vivo

Many microorganisms encode proteins that interact with molecules involved in host immunity; however, few of these molecules have been proven to promote immune evasion in vivo. Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) binds complement component C3 and inhibits complement-mediated virus neutralization and lysis of infected cells in vitro. To investigate the importance of the interaction between gC and C3 in vivo, we studied the virulence of a gC-null strain in complement-intact and C3-deficient animals. Using a vaginal infection model in complement-intact guinea pigs, we showed that gC-null virus grows to lower titers and produces less severe vaginitis than wild-type or gC rescued virus, indicating a role for gC in virulence. To determine the importance of complement, studies were performed with C3-deficient guinea pigs; the results demonstrated significant increases in vaginal titers of gC-null virus, while wild-type and gC rescued viruses showed nonsignificant changes in titers. Similar findings were observed for mice where gC null virus produced significantly less disease than gC rescued virus at the skin inoculation site. Proof that C3 is important was provided by studies of C3 knockout mice, where disease scores of gC-null virus were significantly higher than in complement-intact mice. The results indicate that gC-null virus is approximately 100-fold (2 log10) less virulent that wild-type virus in animals and that gC-C3 interactions are involved in pathogenesis.     

Influence of infectious dose upon productive replication and transynaptic passage of pseudorabies virus in rat central nervous system

Pseudorabies virus (PRV) is a neurotropic swine alpha herpesvirus that characteristically invades the nervous system and replicates within synaptically-linked populations of neurons. The invasive characteristics and ability of this family of viruses to replicate in neurons of the central nervous system (CNS) have been exploited to map functionally related populations of neurons in a variety of systems. In this report, we examined the effects of strain and concentration on the ability of PRV to infect retinal ganglion cells and pass transneuronally through central visual circuits. We find that the ability of virulent (PRV-Becker) and attenuated (PRV-Bartha) strains of PRV to produce a productive infection of visual circuitry is directly dependent upon the infectious of the injected virus. Injections of at least 10(5) total plaque forming units produce 100% infectivity, whereas lower infectious doses substantially reduce the percentage of animals exhibiting productive infection via this route of inoculation. Furthermore, the virulent strain of PRV consistently infects a higher percentage of animals across a broader range of titers than attenuated virus. These data demonstrate that viral titer and strain are important variables that should be considered in the design of studies and interpretation of data derived from investigations employing this pathogen for circuit analysis.     

Radiation-resistant and radiation-sensitive forms of host resistance to polyomavirus

Newborn mice of several inbred strains develop few or no tumors following inoculation with highly tumorigenic strains of polyomavirus. Here we show that such resistant strains can be divided into two groups based on the responses of adult mice to radiation followed by virus inoculation. Most strains show a radiation-sensitive form of resistance (Rr-s) and develop tumors following radiation and virus challenge. This type of resistance has previously been recognized as immunological, based on T-cell responses against virus-encoded neoantigen(s) expressed in tumor cells. Other strains exhibit a radiation-resistant form of resistance (Rr-r) and fail to develop tumors when treated in the same manner. Three additional properties of Rr-r mice distinguish them from Rr-s mice: (i) survival of newborns following inoculation with a highly virulent and usually lethal strain of virus, (ii) resistance to virus spread in newborns inoculated with either tumorigenic or virulent virus strains, and (iii) dominant or semidominant transmission of resistance in crosses with a highly susceptible strain. The Rr-r phenotype reflects a constitutive nonimmunological type of resistance that is targeted to the virus and blocks its dissemination.     

The various Sendai virus C proteins are not functionally equivalent and exert both positive and negative effects on viral RNA accumulation during the course of infection

Recombinant Sendai viruses were prepared which cannot express their Cprime, C, or Cprime plus C proteins due to mutation of their respective start codons ([Cprime-minus], [C-minus] and [double mutant], respectively). The [Cprime-minus] and [C-minus] stocks were similar to that of wild-type (wt) virus in virus titer and plaque formation, whereas the double-mutant stock had a much-reduced PFU or 50% egg infective dose/particle ratio and produced very small plaques. Relative to the wt virus infection, the [Cprime-minus] and [C-minus] infections of BHK cells resulted in significantly greater accumulation of viral RNAs, consistent with the known inhibitory effects of the Cprime and C proteins. The double-mutant infection, in contrast, was delayed in its accumulation of viral RNAs; however, once accumulation started, overaccumulation quickly occurred, as in the single-mutant infections. Our results suggest that the Cprime and C proteins both provide a common positive function early in infection, so that only the double mutant undergoes delayed RNA accumulation and exhibits the highly debilitated phenotype. Later in infection, the same proteins appear to act as inhibitors of RNA accumulation. In infections of mice, [Cprime-minus] was found to be as virulent as wt virus whereas [C-minus] was highly attenuated. These results suggest that the Cprime and C proteins cannot be functionally equivalent, since C can replace Cprime for virulence in mice whereas Cprime cannot replace C.     

Double-labelling immunohistochemical study of megakaryocytes in African swine fever

Bone marrow samples from pigs infected with the highly virulent Malawi'83 or moderately virulent Dominican Republic (DR'78) isolates of African swine fever virus were studied by means of a double labelling immunohistochemical technique which stained the major structural protein VP73 of the virus and megakaryocytes simultaneously. In pigs infected with the highly virulent Malawi'83 isolate, 2.2 per cent of megakaryocytes were VP73+ five days after inoculation, and at six and seven days 2.5 and 9.5 per cent of megakaryocytes were VP73+. Some infected and uninfected megakaryocytes showed pyknosis and karyorrhexis, particularly at seven days after inoculation. However, in comparison with uninfected pigs, the number of megakaryocytes decreased only at seven days after inoculation. In pigs infected with the moderately virulent DR'78 isolate, only 0.2 per cent of megakaryocytes were VP73+ at eight days after inoculation. However, at eight, nine and 10 days after inoculation the total number of megakaryocytes was significantly lower (P < 0.01) than in control uninfected pigs, and the majority of the megakaryocytes showed signs of cell death such as pyknosis and karyorrhexis. The fact that this greater destruction of megakaryocytes was associated with the lower rate of infection of this cell type suggests that indirect damage to megakaryocytes is an additional mechanism of thrombocytopenia in acute and subacute African swine fever.     

Automatic static threshold perimetry is useful for estimating the effects of laser photocoagulation on diabetic maculopathy

Forty-seven Newcastle disease virus (NDV) strains isolated from fecal samples of waterfowls in Alaska and Siberia from 1991 to 1996 were analyzed for their virulence. None of the viruses formed plaques on MDBK cells in the absence of trypsin. Of these, 29 strains showed virulent character by the mean death time with the minimum lethal dose in chicken embryos comparable to velogenic NDV strains. Of the 29 strains, 11 were sequenced for their fusion protein (F) gene. The results showed that 5 of them contained a pair of dibasic amino acids at the cleavage site of the F, which is of a virulent type. The present results suggest that potentially virulent strains of NDV are maintained in migratory waterfowl populations in nature, and that some of those may be transmitted to domestic poultry and acquire pathogenicity during passages in chicken population.     

Resistance to alpha/beta interferons correlates with the epizootic and virulence potential of Venezuelan equine encephalitis viruses and is determined by the 5' noncoding region and glycoproteins

We compared the alpha/beta interferon (IFN-alpha/beta) sensitivities of the TC-83 vaccine strain and 24 enzootic and epizootic Venezuelan equine encephalitis (VEE) isolates. The IFN-resistant or -sensitive phenotype correlated well with epizootic or enzootic potential. IFN-alpha/beta resistance of Trinidad donkey (TRD) virus correlated with virulence determinants in the 5' noncoding region and glycoproteins. Infection of mice lacking a functional IFN system with the IFN-sensitive TC-83 virus resulted in disease equivalent to that produced by the virulent, IFN-resistant TRD virus, further demonstrating that IFN resistance contributes to VEE virus virulence and is a biological marker of epizootic potential.     

Assessment of the MDA and MDMA optical isomers in a stimulant-hallucinogen discrimination

The mild fowlpox vaccine, FPV M, widely used in Australia is composed of two predominant genotypes based upon differences identifiable in restriction enzyme analyses of plaque purified derivatives of this vaccine. The differences, where identifiable, were in the end fragments of the genomes. Five field isolates of FPV from chickens in New South Wales showed restriction enzyme profiles closely related to the more virulent (standard) vaccine strain, FPV S. The FPV S strain differs from FPV M in both terminal genome fragments and in the presence of a PstI fragment of approximately 10kb (this fragment was also present in PstI digests of all of the field isolates). Plaque purified derivatives of FPV M showed similar lesion development upon inoculation into the wing web of chickens. The field isolates showed significantly higher virulence in day-old and three-week-old chickens in comparison with FPV M. One field isolate was similar to the FPV S vaccine. Two isolates had slowly developing wing web lesions, caused significant secondary lesions in three-week-old chickens and generalised poxvirus infection when inoculated into day-old chickens. For two isolates, the primary wing web lesion took even longer to develop and resolve although these isolates did not cause generalised poxvirus infection. It was possible to identify four virulence/pathogenicity types amongst these vaccine and field isolates of FPV. These strains may allow the characterisation of FPV encoded virulence factors. The field strains with higher virulence may be suitable as parent strains for the construction of FPV recombinants with enhanced immune responses to co-expressed vaccine antigens when compared with current FPV M strain based recombinants.     

A model of food-borne Listeria monocytogenes infection in the Sprague-Dawley rat using gastric inoculation: development and effect of gastric acidity on infective dose

Recent epidemiological evidence suggests that Listeria monocytogenes (LM) is a food-borne pathogen in humans. A model of LM infection was developed using the Sprague-Dawley (SD) rat to study the interaction of LM with gastrointestinal epithelium as the first step in the pathogenesis of invasive listeriosis. Conventionally raised, juvenile female SD rats were given 10(2)-10(9) virulent L. monocytogenes, serotype 4b or nonpathogenic Listeria species. Only rats given virulent LM developed dose-dependent invasive infection of the liver and spleen. Light and electron microscopic studies suggested attachment to and invasion of the gastrointestinal mucosa by virulent LM. Because the development of invasive listeriosis in humans has been epidemiologically associated with a decrease in gastric acidity, the effect of decreasing gastric acidity on dose-dependent infection was studied. Rats were pretreated with cimetidine (50 mg/kg) by intraperitoneal injection prior to oral inoculation of 10(2)-10(9) virulent L. monocytogenes. Cimetidine significantly lowered the infective dose of virulent L. monocytogenes (P < 0.05). This oral model should allow further study of host and organism-specific virulence factors mediating the gastrointestinal phase of invasive LM infection, an increasingly important public health problem.     

Immune response and protection in mice inoculated with Leishmania amazonensis clones expressing different degrees of virulence

The induction of protective immunity to Leishmania amazonensis was investigated by injection of parasite clones of low and medium virulence into susceptible mice. To this end, L. amazonensis were cloned by limiting dilution and the clones' virulence was evaluated by the course of infection in susceptible mice. Clones originally derived from the spleen showed virulence variations in comparison with that of the parental population (PP) of parasites. Two low-virulence clones (SP 5 and SP 20) and one medium-virulence clone (SP 11), representative of the spectrum of derived clones, were compared with virulent parasites and with an avirulent strain (Josefa) as to their ability to induce T-cell immune responses and to protect BALB/c mice from infection with the virulent L. amazonensis PP. Clone SP 20 and clone SP 11 induced partial protection when injected by the intravenous and intradermal route, respectively. The avirulent Josefa strain induced neither T-cell responses nor protection. Low-virulence L. amazonensis clones can therefore be additional tools in vaccine investigation.     

Role of the stress-activated protein kinases in endothelin-induced cardiomyocyte hypertrophy

3-beta-Hydroxysteroid dehydrogenase (3-beta-HSD) activity coded for by the A44L gene of vaccinia virus (VV) was demonstrated in CV-1 cultures infected by all five VV strains tested, viz. WR, Praha virus, DRYVAX Wyeth-derived virus (DD), LIVP and MVA. Deletion of the A44L gene in two Praha virus-derived clones (the moderately virulent P13 and the highly attenuated P20), the WR and DD viruses resulted in absence of 3-beta-HSD activity from infected cultures. The virulence for mice of P13 was not affected, and that of WR was only slightly decreased, by the A44L gene deletion. Recombinant VVs expressing either varicella-zoster virus glycoprotein E (VZV-gE) or hepatitis B virus preS2-S protein (HBV-preS2-S) and their respective A44L deleted mutants were used in immunogenicity tests in mice. In terms of antibody response to VV and the recombinant proteins, the deletion resulted in a lowering the immunogenicity in the moderately virulent clone P13 virus and its progenies. In the highly attenuated P20 and DD viruses and their progenies no effects were apparent.     

Construction and characterization of avian Escherichia coli cya crp mutants

We constructed delta cya delta crp mutants of two avian septicemic Escherichia coli strains and evaluated their attenuation in virulence. The P1 phage was used to transfer cya::Tn10 from an E. coli K-12 strain into virulent avian O78 and O2 E. coli isolates. Tetracycline-resistant transductants were plated on Bochner-Maloy Medium, and tetracycline-sensitive colonies were selected, then tested by polymerase chain reaction to confirm that they had deletions of the cya gene. Deletions of crp were created by the same technique in isolates with deletions in cya. The delta cya and delta cya delta crp derivatives had slower growth rates, smaller colonies, and impaired fermentation of carbohydrates compared with their wild parents, and they did not revert. Attenuation of the mutant strains was evaluated by subcutaneous (s.c.) inoculation of day-old chicks and by intratracheal (i.t.) inoculation of 9-day-old chicks previously inoculated intranasally with infectious bronchitis virus. For the wild O78 strain and its delta cya and delta cya delta crp derivatives, the percentages of chicks that died within 6 days of s.c. injection of approximately 5 x 10(7) organisms were 100, 60, and 0, respectively. The corresponding percentages for wild-type O2 and its delta cya and delta cya delta crp mutants were 100, 70, and 20 at a dose of approximately 2 x 10(5) organisms. Following i.t. inoculation, group scores based on pathologic and bacteriologic findings were 51%, 15%, and 9% for wild, delta cya, and delta crp O78 strains (inoculum approximately 2 x 10(7) organisms) and 98%, 31%, and 11%, respectively, for the corresponding O2 strains (inoculum approximately 4 x 10(6) organisms). This study demonstrated reduced virulence and stability of the double mutant, which may useful as a live attenuated vaccine against poultry colibacillosis.     

Inactivation of glycoprotein gE and thymidine kinase or the US3-encoded protein kinase synergistically decreases in vivo replication of pseudorabies virus and the induction of protective immunity

To evaluate the contribution of glycoprotein E (gE), thymidine kinase (TK), and the US3-encoded protein kinase (PK) in the induction of protective immunity to pseudorabies virus (PRV), we intranasally inoculated pigs, the natural host of this virus, with mutant PRV strains in which the genes encoding these proteins were inactivated. Both single and double mutants were constructed. Of these proteins, gE has previously been demonstrated to induce antibodies (in mice and pigs), which require complement to neutralize the virus, and helper T cell responses (in mice). PK and TK have thus far not been reported to induce B or T cell responses. All mutants had a strongly reduced virulence for pigs in comparison with wild-type (wt) PRV. After primary infection, most virus was excreted by wt PRV-inoculated animals. Animals inoculated with gE-PK- and gE-TK- double mutants excreted less virus than animals inoculated with gE-, PK-, and TK- single mutants. After challenge infection with the virulent PRV strain NIA-3, no virus was excreted by wt PRV- and PK- mutant-immunized animals, indicating complete protective immunity. Only one of seven gE- and two of seven TK- mutant-immunized animals excreted virus after the challenge inoculation. In contrast, most animals immunized with the gE-PK- or gE-TK- double mutants excreted virus after the challenge inoculation. Daily mean virus excretion after challenge infection was inversely correlated with daily mean virus excretion after primary infection. In most animals, lack of virus excretion was associated with lack of secondary antibody responses, probably attributable to inadequate stimulation of memory B cells as a consequence of early elimination of viral antigen. Thus, inactivation of gE, TK, and PK all affected the immunogenicity of PRV and the effect of gE and TK and gE and PK inactivation appeared synergistic. We found no simple correlation between in vitro growth properties of the mutants and their immunogenic capacity. Strains lacking PK reached lower end titers in vitro than the other mutants. The most likely explanation for the lower protective capacity of some of the mutants appears their reduced replicative capacity in some cells or tissues in vivo, rather than a loss of particular epitopes.