Mouse hepatitis virus S in weanling Swiss mice following intranasal inoculation

Three-week-old outbred mice were inoculated intranasally with a mildly pathogenic strain of mouse hepatitis virus (MHV-S). Tissues were analyzed for distribution of infectious virus, lesions, and viral antigen at intervals up to 49 days after inoculation. Sera were tested for neutralizing antibody to MHV-S. Within the first week of infection, virus was isolated from lung and brain of most mice and liver of one mouse, but not from blood, spleen, or intestine. Microscopic lesions consisted of mild olfactory mucosal necrosis, neuronal necrosis of olfactory bulbs and tracts, lymphoplasmacytic infiltrates and vacuolation in the brain, mild nonsuppurative pulmonary perivascular lymphocyte infiltration, focal interstitial pneumonia, and focal necrotizing hepatitis. The presence and distribution of MHV antigen, as determined by indirect immunofluorescence, correlated with virus recovery and acute lesions. No virus or antigen was demonstrable beyond day 7. Serum antibody was first detected on day 10, and titers peaked on day 28 after infection.     

Demonstration of vaccinia virus antigen in brains of postvaccinal encephalitis cases.

Five lethal cases of postovaccinal encephalitis (PVE) were examined in attempts to detect the viral antigen by the fluorescent antibody techniques and to isolate the infectious vaccinia virus. Histologically, the brain tissues were characterized by meningoencephalitis, with perivascular infiltrations and inflammatous reactions mainly in leptomininx, chroid plexus and ependyma. By direct immunofluorescence staining, vaccinia virus antigen was found in the brain specimens of one case, but not in the other four cases. The existence of antigen-antibody complexes was suggested by the deposition of immunoglobulins in the brain specimens. After treatment with 3 M NaSCN solution to dissociate the immunoglobulins from the complex, the viral antigen was demonstrated in specimens of two cases. Localization of viral antigen(s) thus detected coincided with that of meningoencephalitic reactions in the histological examination. Virus isolation on chick embryo chorioallantoic membrane was negative in all cases. Participation of viral pathogenicity in the etiology of PVE is discussed.     

Production of a particulate hepatitis C vaccine candidate by an engineered Lactococcus lactis strain

Vaccine delivery systems based on display of antigens on bioengineered bacterial polyester inclusions can stimulate cellular immune responses. The food-grade Gram-positive bacterium Lactococcus lactis was engineered to produce spherical polyhydroxybutyrate (PHB) inclusions which abundantly displayed the hepatitis C virus core (HCc) antigen. In mice, the immune response induced by this antigen delivery system was compared to that induced by vaccination with HCc antigen displayed on PHB beads produced in Escherichia coli, to PHB beads without antigen produced in L. lactis or E. coli, or directly to the recombinant HCc protein. Vaccination site lesions were minimal in all mice vaccinated with HCc PHB beads or recombinant protein, all mixed in the oil-in-water adjuvant Emulsigen, while vaccination with the recombinant protein in complete Freund's adjuvant produced a marked inflammatory reaction at the vaccination site. Vaccination with the PHB beads produced in L. lactis and displaying HCc antigen produced antigen-specific cellular immune responses with significant release of gamma interferon (IFN-γ) and interleukin-17A (IL-17A) from splenocyte cultures and no significant antigen-specific serum antibody, while the PHB beads displaying HCc but produced in E. coli released IFN-γ and IL-17A as well as the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-6 and low levels of IgG2c antibody. In contrast, recombinant HCc antigen in Emulsigen produced a diverse cytokine response and a strong IgG1 antibody response. Overall it was shown that L. lactis can be used to produce immunogenic PHB beads displaying viral antigens, making the beads suitable for vaccination against viral infections.     

Epstein-Barr病毒基因工程活疫苗人体免疫的初步研究

Epstein-Barr(EB)病毒的原发感染发生在儿童时期,在我国3～5岁儿童的感染率为70％～90％。感染后终生带毒,并经唾液不断排出病毒。我国南方是鼻咽癌高发区,其发病率和死亡率均占恶性肿瘤的第一位。早期诊断方法的改进和早期治疗,使鼻咽癌治疗后的5年生存率明显增加,但不能降低发病率。EB病毒疫苗有可能成为控制该病的有效手段之一。 Epstein等人从淋巴母细胞株(B95-8细胞)细胞表面提取EB病毒膜抗原(MA),用于免疫棉顶猴能产生中和抗体。免疫动物能抵抗EB病毒攻击后所诱发的恶性淋巴瘤。该中和抗体在体外能中和EB病毒的转化活性。EB病毒的主要膜抗原(MA)是由分子量220kD和     

Pathogenicity of a vaccine strain of vaccina virus in rabbits administered by intravenous route.

The pathogenicity of the Lister (Elstree) strain, a vaccinia virus, in rabbits after intravenous injection was studied by histopathological and immunofluorescent methods. Inoculation of 1 X 10(7) PFU (pock-forming unit) virus into weanling and young adult rabbits caused severe emaciation and high mortality within 2 weeks. Pathological findings were characterized by vesicular lesions along muco-cutaneous junction areas of the eyes, nose and mouth and by inflammatory changes in the brain, mainly in the meninges and choroid plexus. Immunofluorescent staining of the tissues of animals sacrificed at intervals demonstrated the accumulation of vaccinia viral antigen (s) in the loci of pathological changes. The suitability of this model system for the study of pathogenesis of human postvaccinal meningoencephalitis is discussed.     

Lesions and tissue distribution of viral antigen in severe acute versus subclinical acute infection with BVDV2.

Differences in the distribution and spread of viral antigen, development of lesions and correlation between presence of viral antigen and lesions were compared between a low and highly virulent strain of BVDV2. Two groups of two-week- to two-month-old colostrum-deprived calves were inoculated intranasally with the naturally occurring low virulent BVDV2 strain 28508-5 or the highly virulent strain 1373. To study the sequence of virus spread and lesion development, calves were necropsied at days three, six, eight-nine and 12 to 14 post inoculation (pi). Viral antigen was detected by the indirect immunoperoxidase method in cryostat sections and lesions were evaluated in H&E-stained paraffin sections. Clinical signs and changes in lymphocyte and thrombocyte numbers confirmed the difference in virulence between the two strains. Both strains showed comparable initial infection and spread at day three pi. At day six pi, they were found widespread in lymphoid tissues and multifocally in intestinal mucosa. Lesions were very mild despite the large amount of antigen in the lymphoid tissues. After day six pi, differences between the low and highly virulent strains became more prominent. The strain of low virulence was cleared from the tissues, but there was a transient phase of depletion. The highly virulent strain continued to spread to different organs and there was severe depletion of lymphoid tissues without recovery.     

Vaccinia virus persistence in a child against the background of immune deficiency

A young girl, vaccinated against smallpox 6 years before suffered from a persistent vaccinia virus infection and a congenital skin disease, i.e. epidermolysis bullosa. The virus was isolated from skin lesions at the vaccination site and remote sites and repeatedly from the blood; it was not isolated from bone-marrow specimen, saliva, pharynx or urine. The titre of virus-neutralizing antibodies was low (1:10), immunoglobulins A, M and G were within age-related limits; antibodies against measles and tetanus were at protective levels, skin tests were positive. Staphylococcal antitoxin titre was extremely high. The child's mother, not vaccinated against smallpox, possessed vaccinia--virus-neutralizing antibodies at high titre (over 1:320). Examination of the child did not show any quantitative immune deficiency. Immune deviations were found in the lymphocyte blast-transformation reaction on stimulation with PHA and specific antigen, as well as in the nonspecific-suppression test. The possible genesis of the virus persistence and the role of the virus in the clinical course of the disease are discussed.     

Yaba tumor poxvirus synthesis in vitro. I. Cytopathological, histochemical, and immunofluorescent studies

Yohn, David S. (Roswell Park Memorial Institute, Buffalo, N.Y.), Victoria A. Haendiges, and James T. Grace, Jr. Yaba tumor poxvirus synthesis in vitro. I. Cytopathological, histochemical, and immunofluorescent studies. J. Bacteriol. 91:1977-1985. 1966.-Yaba tumor poxvirus synthesis in BSC-1 cell culture was followed sequentially with light microscopy, immunofluorescent microscopy, and various histochemical stains. The first evidence of infection was detected at 24 hr when nucleoli became hypertrophic, reflecting enhanced ribonucleic acid (RNA) synthesis. At 36 hr, deoxyribonucleic acid synthesis was detected in the cytoplasm. This was immediately followed by or associated with antigen synthesis at paranuclear sites and enhanced RNA synthesis in the cytoplasm. Cytoplasmic inclusions were readily apparent at 4 days in initially infected cells. Contiguous spread of virus was judged to have occurred around the third day of infection. The time required to complete the synthetic cycle from time of infection to production of infectious progeny was estimated to be of the order of 60 hr. This cycle is 6 to 10 times longer than for vaccinia virus. By light microscopy, cytopathic effects (CPE) were detectable in 5 days in heavily infected cultures. With 100 units or less of infectious virus, CPE was not readily detected until the 10th to 14th day. At this time, focal areas of infection classified either as microtumors or microplaques were present. Secondary foci appeared during the 4th week of incubation.     

Participation of vaccinia virus in the pathogenesis of different clinical forms of postvaccinal complications. I. Frequency of vaccinia virus detection in the vaccinted who have usual and complicated reactions to vaccination]

The virological examination of 1365 samples taken from 469 children vaccinated against smallpox revealed considerable differences in the frequency and the time of vaccinia virus detection in different clinical forms of postvaccinal pathology as compared with uncomplicated vaccinal process. During the postvaccinal period taking its normal course vaccinia virus was isolated from 7.3% of children only from the pharynx till day 8 following vaccination. In generalized and creeping vaccinia the virus was isolated from 71.4% of children, in postvaccinal encephalitis from 57.1% of children, in vaccinal angina frove-mentioned complications vaccinia virus was detected in the samples obtained from the patients till days 24, 35, 15 and 24 respectively. The etiopathogenetic role of vaccinia virus in a number of postvaccinal complications is discussed.     

Pathogenesis of mouse hepatitis virus infection. The role of nasal epithelial cells as a primary target of low-virulence virus, MHV-S.

The pathogenesis of mouse hepatitis virus (MHV-S) infection in suckling and weanling mice was comparatively studied after intranasal inoculation. In sucklings, infectious virus as well as specific antigen was first detected in the nasal mucosa at 12 hr, then in the nerve cells of the olfactory bulbs. At this stage viral particles were demonstrated both in the supporting cells and olfactory cells of the nasal mucosa. In the posterior part of the brain and spinal cord, virus was detected on days 3 to 4 postinoculation when viral growth was clearly demonstrable in the liver, spleen and intestines. In weanlings too, infection was first established in the nasal mucosa, shedding infectious virus in the nasal washing until day 6 postinoculation, and later infection spread to the brain and spinal cord. In weanling mice, however, neither infectious virus nor viral antigen was detected in the liver or other visceral organs, while serum neutralizing antibody became detectable on day 5 postinoculation, increasing in titer thereafter. Histopathologically degenerative and necrotic changes were observed in the nasal mucosa and central nervous system of both age groups of animals coincidentally with the presence of viral specific antigen, while inflammatory response was much less prominent in sucklings. In the liver, spleen and intestines, however, some lesions were observed only in sucklings.     

Immune Response in Goats to Different Payloads of FMDV Monovalent Vaccine: Protection Against Virulent Challenge and Development of Carrier Status

The relationship of Foot-and-Mouth Disease virus antigen payload and number of dose of vaccine conferring protection against virus challenge in goats was studied. Goats vaccinated with oil adjuvant Foot-and-Mouth Disease vaccines containing different antigen payloads with or without booster resisted virulent challenge at 21 days post-vaccination or 7 days after booster respectively. However, localized sub-clinical infection was observed in two vaccinated goats on 35 days post-challenge. RNA could be detected from 31.8% of vaccinated goats (10^2.69–10^4.99 viral RNA copies per cotton swab of nasal secretions) on day 35 post-challenge. Since no live virus could be isolated after 5 days post-challenge, the risk of these animals transmitting the disease was probably very low. The finding showed that oil adjuvant Foot-and-Mouth Disease vaccines containing antigen payload of 1.88 μg may prevent or reduce the local virus replication at the oropharynx and shedding of virus from nasal secretions and thereby reduce the amount of virus released into the environment subsequent to exposure to live virus. This study also showed that goats with poor sero conversion to vaccination can be infected without overt clinical signs and became carriers like sheep.     

Passive immunotherapies protect WRvFire and IHD-J-Luc vaccinia virus-infected mice from lethality by reducing viral loads in the upper respiratory tract and internal organs

Whole-body bioimaging was employed to study the effects of passive immunotherapies on lethality and viral dissemination in BALB/c mice challenged with recombinant vaccinia viruses expressing luciferase. WRvFire and IHD-J-Luc vaccinia viruses induced lethality with similar times to death following intranasal infection, but WRvFire replicated at higher levels than IHD-J-Luc in the upper and lower respiratory tracts. Three types of therapies were tested: licensed human anti-vaccinia virus immunoglobulin intravenous (VIGIV); recombinant anti-vaccinia virus immunoglobulin (rVIG; Symphogen, Denmark), an investigational product containing a mixture of 26 human monoclonal antibodies (HuMAbs) against mature virion (MV) and enveloped virion (EV); and HuMAb compositions targeting subsets of MV or EV proteins. Bioluminescence recorded daily showed that pretreatment with VIGIV (30 mg) or with rVIG (100 μg) on day -2 protected mice from death but did not prevent viral replication at the site of inoculation and dissemination to internal organs. Compositions containing HuMAbs against MV or EV proteins were protective in both infection models at 100 μg per animal, but at 30 μg, only anti-EV antibodies conferred protection. Importantly, the t statistic of the mean total fluxes revealed that viral loads in surviving mice were significantly reduced in at least 3 sites for 3 consecutive days (days 3 to 5) postchallenge, while significant reduction for 1 or 2 days in any individual site did not confer protection. Our data suggest that reduction of viral replication at multiple sites, including respiratory tract, spleen, and liver, as monitored by whole-body bioluminescence can be used to predict the effectiveness of passive immunotherapies in mouse models.     

Persistence of sialodacryoadenitis virus in athymic rats

To determine whether SDAV infection persists in athymic rats, weanling athymic rats and euthymic rats were inoculated intranasally with 10(4) TCID50 of SDAV and examined periodically for up to 90 days. Viral antigen and lesions characteristic of acute SDAV infection, including rhinotracheitis, bronchitis and sialodacryoadenitis, were detected in both groups of rats during the first week. In euthymic rats, tissues were under repair and viral antigen was undetectable by day 17, and tissues were histologically normal by day 31 except for mild focal dacryoadenitis. In athymic rats, viral antigen and chronic active inflammation of respiratory tract, salivary and lacrimal glands persisted through day 90. Inflammation and viral antigen also were observed in the transitional epithelium of the renal pelvis and urinary bladder as late as day 90. Virus was isolated from nasopharynx, lung, salivary gland and Harderian gland of athymic rats through day 90. All euthymic rats seroconverted to SDAV by day 6, whereas all athymic rats remained seronegative through day 31, and two of six were seropositive by day 90. As judged by seroconversion of contact sentinels, six of six athymic rats shed virus through 6 weeks, and five of six through 10 weeks. These results indicate that SDAV persists in athymic rats, and that normal T cell function is required for host defenses against SDAV.     

Mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. II. Pathogenesis

The pathogenesis of mousepox due to infection with ectromelia virus strain NIH-79 was characterized in genetically susceptible (BALB/cAnNCr) and genetically resistant (C57BL/6NCr) mice. BALB/c mice inoculated subcutaneous (s.c.) or intranasally (i.n.) had high mortality. Most mice died within 7 days from severe necrosis of the spleen and liver. Necrotic foci in livers of BALB/c mice that survived beyond 7 days often were accompanied by mononuclear cell infiltrates and by hyperplasia of lymphoid tissues. C57BL/6 mice inoculated by either route remained asymptomatic and necrotic lesions were mild or absent, whereas focal non-suppurative hepatitis and lymphoid hyperplasia were prominent. Infectious virus and viral antigen were distributed widely in tissues of BALB/c mice, but had limited distribution in C57BL/6 mice. Both mouse strains had infection of the respiratory tract, genital tract, oral tissues and bone marrow, and BALB/c mice also had infection of the intestines. Both strains also developed serum antibody to vaccinia virus antigen after infection. The results show that ectromelia virus occurs in tissues conducive to mouse to mouse transmission and that the severity and character of mousepox lesions correlate directly with resistance and susceptibility to infection. They also support the concept that cellular immunity contributes to survival from infection.     

Development of a Genus-Specific Antigen Capture ELISA for Orthopoxviruses – Target Selection and Optimized Screening

Orthopoxvirus species like cowpox, vaccinia and monkeypox virus cause zoonotic infections in humans worldwide. Infections often occur in rural areas lacking proper diagnostic infrastructure as exemplified by monkeypox, which is endemic in Western and Central Africa. While PCR detection requires demanding equipment and is restricted to genome detection, the evidence of virus particles can complement or replace PCR. Therefore, an easily distributable and manageable antigen capture enzyme-linked immunosorbent assay (ELISA) for the detection of orthopoxviruses was developed to facilitate particle detection. By comparing the virus particle binding properties of polyclonal antibodies developed against surface-exposed attachment or fusion proteins, the surface protein A27 was found to be a well-bound, highly immunogenic and exposed target for antibodies aiming at virus particle detection. Subsequently, eight monoclonal anti-A27 antibodies were generated and characterized by peptide epitope mapping and surface plasmon resonance measurements. All antibodies were found to bind with high affinity to two epitopes at the heparin binding site of A27, toward either the N- or C-terminal of the crucial KKEP-segment of A27. Two antibodies recognizing different epitopes were implemented in an antigen capture ELISA. Validation showed robust detection of virus particles from 11 different orthopoxvirus isolates pathogenic to humans, with the exception of MVA, which is apathogenic to humans. Most orthopoxviruses could be detected reliably for viral loads above 1 × 10³ PFU/mL. To our knowledge, this is the first solely monoclonal and therefore reproducible antibody-based antigen capture ELISA able to detect all human pathogenic orthopoxviruses including monkeypox virus, except variola virus which was not included. Therefore, the newly developed antibody-based assay represents important progress towards feasible particle detection of this important genus of viruses.     

Subacute sclerosing panencephalitis in Croatia (1994-2004)

We studied five patients with SSPE during a 10-year period (1994-2004). The first clinical symptoms developed at the age of 5-11 years. All patients were vaccinated regularly against measles according to the official immunization schedule. One patient had measles at the age of 18 months. Two of them had a history of morbilliform rash (unrecognized measles) at the age of six and seven months, respectively. In two patients, with no history of measles before vaccination the disease started after varicella infection. Using complement-fixation (F) test and EIA, antibodies to measles virus (MV) were detected in the CSF and sera of all patients. The CF-antibody titers ranged from 1:1024 to 1:65536 in sera and from 1:16 to 1:128 in CSF samples. MV antigen was detected in brain imprints using IFA in two patients. Electron microscopic analysis revealed intranuclear viral inclusions (MV nucleocapsids). Using RT-PCR, viral RNA was found in both patients. Nucleotide sequence analysis showed that the viruses found in the brain tissue belonged to the wild-type MV D6 genotype [7].     

Editing of the Sendai virus P/C mRNA by G insertion occurs during mRNA synthesis via a virus-encoded activity.

Two forms of the Sendai virus P/C mRNA have been predicted: one an exact copy of the viral genome, and the other with a single G insertion within a run of three G's. We directly cloned the mRNA or portions of it containing the insertion site and screened the resulting colonies with oligonucleotides that could distinguish the presence of three or four G's at this position. We found that 31% of the mRNAs did in fact contain the predicted insertion, whereas the viral genomes contained no heterogeneity at this position. A smaller fraction (7%) of the mRNA contained two to eight G's inserted at this position. The insertions also took place during RNA synthesis in vitro with purified virions but were not detected when the mRNA was expressed in vivo via a vaccinia virus recombinant. When the Sendai virus- and vaccinia virus-derived P/C mRNAs were coexpressed in the same cells under conditions in which each could be distinguished, those from the Sendai genome were altered as before, but those from the vaccinia virus genome remained unaltered. The activity that alters the mRNA is therefore likely to be coded for by the virus and cannot function in trans.     

Murine infection by vesicular stomatitis virus: initial characterization of the H-2d system.

BALB/c mice and congenic H-2Ld-deficient BALB/c-H-2dm2 (dm2) mice were experimentally infected intranasally with isolates of vesicular stomatitis virus (VSV). The survival of infected hosts, viral replication in lungs and brains, and histopathologic in the two mouse strains were compared. In both strains of mice, mortality occurred during the period 7 to 10 days postinfection. However, dm2 mice were relatively resistant to lethal infections. Viral replication occurred at low levels in the lungs of both strains and did not evoke significant pathologic changes. In contrast, viral replication in the brains was much greater; in the BALB/c strain, this was accompanied by more frequent and more severe pathologic changes. In general, mice surviving at day 10 had effectively cleared virus from central nervous system but not respiratory sites. Evidence is presented that viral replication occurs first in the nasal cavity and is transmitted both to the lungs and to the olfactory bulb where focal cytopathology occurs. Virus enters the ventricles, causing encephalitis; necrosis occurs around the ventricles and in the lumbosacral region of the spinal cord. Necrotic lesions were accompanied by mononuclear infiltration. Mice immunized with virus of the same serotype or with a vaccinia virus hybrid encoding the VSV glycoprotein were protected from lethal infection; in contrast, mice immunized with heterotypic virus were susceptible to challenge.     

Immune modulation by IL-10 gene transfer via viral vector and plasmid DNA: implication for gene therapy.

In the present report, we have evaluated and compared the modulatory effect of the cytokine interleukin (IL)-10 expressed via viral vector or plasmid DNA on viral antigen-induced cutaneous inflammatory lesions. Our data demonstrate the superior potency of both recombinant vaccinia virus and herpes simplex virus IL-10 expression vectors after single intramuscular administration, but the effects were only short term and only functioned in animals lacking immunity to the viral vectors used for modulation. In contrast, modulatory effects achieved by plasmid DNA expressing IL-10 were delayed in onset and milder in effect but were far more persistent than those achieved by viral vectors. Moreover, plasmid DNA expressing IL-10 provided effective modulation when given repeatedly to animals. Our data also showed that IL-10 gene delivery resulted in a systemic and durable modulatory effect while the effect caused by a single IL-10 protein treatment was transient and confined to the injected site. Our results imply that the viral vector system is superior for obtaining short-term effects, whereas the plasmid DNA approach represents a better strategy to achieve gene therapy to modulate chronic inflammatory lesions.     

Long-Term Sterilizing Immunity to Rinderpest in Cattle Vaccinated with a Recombinant Vaccinia Virus Expressing High Levels of the Fusion and Hemagglutinin Glycoproteins

Rinderpest is an acute and highly contagious viral disease of ruminants, often resulting in greater than 90% mortality. We have constructed a recombinant vaccinia virus vaccine (v2RVFH) that expresses both the fusion (F) and hemagglutinin (H) genes of rinderpest virus (RPV) under strong synthetic vaccinia virus promoters. v2RVFH-infected cells express high levels of the F and H glycoproteins and show extensive syncytium formation. Cattle vaccinated intramuscularly with as little as 10(3) PFU of v2RVFH and challenged 1 month later with a lethal dose of RPV were completely protected from clinical disease; the 50% protective dose was determined to be 10(2) PFU. Animals vaccinated with v2RVFH did not develop pock lesions and did not transmit the recombinant vaccinia virus to contact animals. Intramuscular vaccination of cattle with 10(8) PFU of v2RVFH provided long-term sterilizing immunity against rinderpest. In addition to being highly safe and efficacious, v2RVFH is a heat-stable, inexpensive, and easily administered vaccine that allows the serological differentiation between vaccinated and naturally infected animals. Consequently, mass vaccination of cattle with v2RVFH could eradicate rinderpest.