The Effect of Epstein-Barr Virus Latent Membrane Protein 2 Expression on the Kinetics of Early B Cell Infection

Infection of human B cells with wild-type Epstein-Barr virus (EBV) in vitro leads to activation and proliferation that result in efficient production of lymphoblastoid cell lines (LCLs). Latent Membrane Protein 2 (LMP2) is expressed early after infection and previous research has suggested a possible role in this process. Therefore, we generated recombinant EBV with knockouts of either or both protein isoforms, LMP2A and LMP2B (Δ2A, Δ2B, Δ2A/Δ2B) to study the effect of LMP2 in early B cell infection. Infection of B cells with Δ2A and Δ2A/Δ2B viruses led to a marked decrease in activation and proliferation relative to wild-type (wt) viruses, and resulted in higher percentages of apoptotic B cells. Δ2B virus infection showed activation levels comparable to wt, but fewer numbers of proliferating B cells. Early B cell infection with wt, Δ2A and Δ2B viruses did not result in changes in latent gene expression, with the exception of elevated LMP2B transcript in Δ2A virus infection. Infection with Δ2A and Δ2B viruses did not affect viral latency, determined by changes in LMP1/Zebra expression following BCR stimulation. However, BCR stimulation of Δ2A/Δ2B cells resulted in decreased LMP1 expression, which suggests loss of stability in viral latency. Long-term outgrowth assays revealed that LMP2A, but not LMP2B, is critical for efficient long-term growth of B cells in vitro. The lowest levels of activation, proliferation, and LCL formation were observed when both isoforms were deleted. These results suggest that LMP2A appears to be critical for efficient activation, proliferation and survival of EBV-infected B cells at early times after infection, which impacts the efficient long-term growth of B cells in culture. In contrast, LMP2B did not appear to play a significant role in these processes, and long-term growth of infected B cells was not affected by the absence of this protein. © 2013 Wasil et al

Deathly Drool: Evolutionary and Ecological Basis of Septic Bacteria in Komodo Dragon Mouths

Komodo dragons, the world's largest lizard, dispatch their large ungulate prey by biting and tearing flesh. If a prey escapes, oral bacteria inoculated into the wound reputedly induce a sepsis that augments later prey capture by the same or other lizards. However, the ecological and evolutionary basis of sepsis in Komodo prey acquisition is controversial. Two models have been proposed. The “bacteria as venom” model postulates that the oral flora directly benefits the lizard in prey capture irrespective of any benefit to the bacteria. The “passive acquisition” model is that the oral flora of lizards reflects the bacteria found in carrion and sick prey, with no relevance to the ability to induce sepsis in subsequent prey. A third model is proposed and analyzed here, the “lizard-lizard epidemic” model. In this model, bacteria are spread indirectly from one lizard mouth to another. Prey escaping an initial attack act as vectors in infecting new lizards. This model requires specific life history characteristics and ways to refute the model based on these characteristics are proposed and tested. Dragon life histories (some details of which are reported here) prove remarkably consistent with the model, especially that multiple, unrelated lizards feed communally on large carcasses and that escaping, wounded prey are ultimately fed on by other lizards. The identities and evolutionary histories of bacteria in the oral flora may yield the most useful additional insights for further testing the epidemic model and can now be obtained with new technologies

Efficacy of topical cobalt chelate CTC-96 against adenovirus in a cell culture model and against adenovirus keratoconjunctivitis in a rabbit model

BACKGROUND: Adenovirus (Ad), associated with significant morbidity, has no topical treatment. A leading CTC compound (CTC-96), a Co(III )chelate, was found to have potent in vitro and in vivo antiviral efficacy against herpes viruses. In this study CTC-96 is being tested for possible anti-Adenovirus activity. METHODS: The biological anti-adenovirus activity of CTC-96 in concentrations from 5 to 250 ug/ml, was evaluated initially by viral inactivation (viral exposure to CTC-96 followed by dilution and inoculation of cells), virucidal (viral exposure to CTC-96 and inoculation of cells without dilution) and antiviral (effect of CTC-96 on previously adsorbed virus) plaque assays on HeLa (human cervical carcinoma), A549 (human lung carcinoma) and SIRC (rabbit corneal) cells. After verifying the antiviral activity, New Zealand White rabbits were infected with Ad-5 into: 1) the anterior cul-de-sac scarifying the conjunctiva (Group "C+"); 2) the anterior cul-de-sac scarifying the conjunctiva and cornea (Group "CC+"); 3) the stroma (Group "CI+"). Controls were sham-infected ("C-", "CC-", "CI-"). Other rabbits, after "CC", were treated for 21 days with: 1) placebo, 9x/day ("-"); 2) CTC-96, 50 ug/ml, 9x/day ("50/9"); CTC-96, 50 ug/ml, 6x/day ("50/6"); CTC-96, 25 ug/ml, 6x/day ("25/6"). All animals were monitored via examination and plaque assays. RESULTS: In vitro viral inactivation, virucidal and antiviral assays all demonstrated CTC-96 to be effective against Adenvirus type 5 (ad-5). The in vivo model of Ad keratoconjunctivitis most similar to human disease and producing highest viral yield was "CC". All eyes (6/6) developed acute conjunctivitis. "CI" yielded more stromal involvement (1/6) and iritis (5/6), but lower clinical scores (area × severity). Infection via "C" was inconsistent (4/6). Fifty (50) ug/ml was effective against Ad-5 at 6x, 9x dosings while 25 ug/ml (6x) was only marginally effective. CONCLUSION: CTC-96 demonstrated virucidal activity against Ad5 in tissue culture with HeLa, A549 and SIRC cell lines. Animal Model Development: 1) "CC" produced conjunctival infection with occasional keratitis similar to human disease; "CI" yielded primarily stromal involvement; 2) "C" consistently produced neither conjunctivitis nor keratitis. CTC Testing: 1) Conjunctivitis in all eyes; 2) Resolution fastest in "50/9" ("50/9". "50/6" > "25/6" > "-"); 3) Efficacy in "50/6" was not statistically different than "50/9"; 4) Conjunctival severity was lower in treatment groups then controls; 5) Little corneal or intra-ocular changes were noted

Development of real-time NASBA assays with molecular beacon detection to quantify mRNA coding for HHV-8 lytic and latent genes

BACKGROUND: Human herpesvirus-8 (HHV-8) is linked to the pathogenesis of Kaposi's sarcoma (KS), and the HHV-8 DNA load in peripheral blood mononuclear cells (PBMC) is associated with the clinical stage of KS. To examine the expression of HHV-8 in PBMC, four HHV-8 mRNA specific NASBA assays were developed METHODS: We have developed four quantitative nucleic acid sequence-based amplification assays (NASBA-QT) specifically to detect mRNA coding for ORF 73 (latency-associated nuclear antigen, LANA), vGCR (a membrane receptor), vBcl-2 (a viral inhibitor of apoptosis) and vIL-6 (a viral growth factor). The NASBA technique amplifies nucleic acids without thermocycling and mRNA can be amplified in a dsDNA background. A molecular beacon is used during amplification to enable real-time detection of the product. The assays were tested on PBMC samples of two AIDS-KS patients from the Amsterdam Cohort. RESULTS: For all four assays, the limit of detection (LOD) of 50 molecules and the limit of quantification (LOQ) of 100 molecules were determined using in vitro transcribed RNA. The linear dynamic range was 50 to 10(7) molecules of HHV-8 mRNA. We found HHV-8 mRNA expression in 9 out of the 10 tested samples. CONCLUSION: These real-time NASBA assays with beacon detection provide tools for further study of HHV-8 expression in patient material

Aspects of the encephalitis epidemic caused by arbovirus in the Ribeira Valley, S. Paulo, Brazil, during 1975-1978

In 1975, 1976, and 1977, arbovirus caused an encephalitis epidemic in the Ribeira Valley in the state of S. Paulo. The epidemic would peak when the temperature and pluvial levels were higher. From 1978 on the disease maintained low levels within a presumed endemic zone. The epidemic had swept from east to west and from east to southwest in a wave toward the neighboring coastal region. The mountain chain to the north and northwest acted as a barrier. It was only logical natural that the hypothesis that the etiological agent, arbovirus Rocio may have recently infected the human population be considered. Mosquitos must have transmitted the infection from birds and small mammals in nearby forests. Probable forms of transmission of arboviroses in the home are also discussed in this article as well as the fact that population groups that presented the worst forms of the disease were the very young, and the very old and those living in the worst conditions. The epidemiological perspective of the arboviroses is that it persists in this area because the area presents excellent conditions for the development of the etiological agent - reservoirs and biological vectors, with a continuous supply of susceptible people, migrants or tourists.Foi realizado estudo epidemiológico da encefalite por arbovirus na região do Vale do Ribeira, S. Paulo, Brasil. Uma epidemia da moléstia ocorreu em 1975, 1976 e 1977, com picos nas épocas de maior temperatura e pluviosidade. A partir de 1978 a moléstia manteve-se em níveis baixos numa presumível ende-micidade. A epidemia se deslocou em onda em direção leste-oeste e leste-sudoeste para a região litorânea vizinha. A cadeia montanhosa situada ao norte e noroeste atuou como barreira à propagação da moléstia. Considerou-se a hipótese que o agente etiológico, arbovirus Rocio, deva ter começado a infectar a população humana recentemente, tendo sido veiculado ao homem de reservatórios silvestres, aves e pequenos mamíferos, por culicídeos silvestres. Discutiu-se também prováveis formas de transmissão domiciliar. Verificou-se que os grupos populacionais que apresentaram as formas mais graves foram os de idades extremas e os que apresentavam piores condições de vida. Considerou-se que a perspectiva epidemiológica desta arbovirose é que ela persista na região, uma vez que a mesma apresenta condições ótimas para o desenvolvimento do agente etiológico, dos reservatórios e dos vetores biológicos, além de receber um contínuo afluxo de população suscetível, migrantes ou turistas

Serologic evidence of the recent circulation of Saint Louis encephalitis virus and high prevalence of equine encephalitis viruses in horses in the Nhecolândia sub-region in South Pantanal, Central-West Brazil

As in humans, sub-clinical infection by arboviruses in domestic animals is common; however, its detection only occurs during epizootics and the silent circulation of some arboviruses may remain undetected. The objective of the present paper was to assess the current circulation of arboviruses in the Nhecolândia sub-region of South Pantanal, Brazil. Sera from a total of 135 horses, of which 75 were immunized with bivalent vaccine composed of inactive Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus(WEEV) and 60 were unvaccinated, were submitted to thorough viral isolation, reverse transcriptase polymerase chain reaction (RT-PCR) and neutralization tests for Saint Louis encephalitis virus (SLEV), EEEV, WEEV and Mayaro virus (MAYV). No virus was isolated and viral nucleic-acid detection by RT-PCR was also negative. Nevertheless, the prevalence of neutralizing antibodies in horses older than seven months was 43.7% for SLEV in equines regardless of vaccine status, and 36.4% for WEEV and 47.7% for EEEV in unvaccinated horses. There was no evidence of MAYV infections. The serologic evidence of circulation of arboviruses responsible for equine and human encephalitis, without recent official reports of clinical infections in the area, suggests that the Nhecolândia sub-region in South Pantanal is an important area for detection of silent activity of arboviruses in Brazil

Persistence of experimental Rocio virus infection in the golden hamster (Mesocricetus auratus)

Rocio virus (ROCV) is an encephalitic flavivirus endemic to Brazil. Experimental flavivirus infections have previously demonstrated a persistent infection and, in this study, we investigated the persistence of ROCV infection in golden hamsters (Mesocricetus auratus). The hamsters were infected intraperitoneally with 9.8 LD50/0.02 mL of ROCV and later anaesthetised and sacrificed at various time points over a 120-day period to collect of blood, urine and organ samples. The viral titres were quantified by real-time-polymerase chain reaction (qRT-PCR). The specimens were used to infect Vero cells and ROCV antigens in the cells were detected by immunefluorescence assay. The levels of antibodies were determined by the haemagglutination inhibition technique. A histopathological examination was performed on the tissues by staining with haematoxylin-eosin and detecting viral antigens by immunohistochemistry (IHC). ROCV induced a strong immune response and was pathogenic in hamsters through neuroinvasion. ROCV was recovered from Vero cells exposed to samples from the viscera, brain, blood, serum and urine and was detected by qRT-PCR in the brain, liver and blood for three months after infection. ROCV induced histopathological changes and the expression of viral antigens, which were detected by IHC in the liver, kidney, lung and brain up to four months after infection. These findings show that ROCV is pathogenic to golden hamsters and has the capacity to cause persistent infection in animals after intraperitoneal infection