Early and late neuropathological features of meningoencephalitis associated with Maraba virus infection

Maraba virus is a member of the genus Vesiculovirus of the Rhabdoviridae family that was isolated in 1983 from sandflies captured in the municipality of Maraba, state of Pará, Amazônia, Brazil. Despite 30 years having passed since its isolation, little is known about the neuropathology induced by the Maraba virus. Accordingly, in this study the histopathological features, inflammatory glial changes, cytokine concentrations, and nitric oxide activity in the encephalon of adult mice subjected to Maraba virus nostril infection were evaluated. The results showed that 6 days after intranasal inoculation, severe neuropathological-associated disease signs appeared, including edema, necrosis and pyknosis of neurons, generalized congestion of encephalic vessels, and intra- and perivascular meningeal lymphocytic infiltrates in several brain regions. Immunolabeling of viral antigens was observed in almost all central nervous system (CNS) areas and this was associated with intense microglial activation and astrogliosis. Compared to control animals, infected mice showed significant increases in interleukin (IL)-6, tumor necrosis factor (TNF)-α, interferon (INF)-γ, MCP-1, nitric oxide, and encephalic cytokine levels. We suggest that an exacerbated inflammatory response in several regions of the CNS of adult BALB/c mice might be responsible for their deaths.

A real-time reverse transcriptase polymerase chain reaction for detection and quantification of Vesiculovirus

Vesiculoviruses (VSV) are zoonotic viruses that cause vesicular stomatitis disease in cattle, horses and pigs, as well as sporadic human cases of acute febrile illness. Therefore, diagnosis of VSV infections by reliable laboratory techniques is important to allow a proper case management and implementation of strategies for the containment of virus spread. We show here a sensitive and reproducible real-time reverse transcriptase polymerase chain reaction (RT-PCR) for detection and quantification of VSV. The assay was evaluated with arthropods and serum samples obtained from horses, cattle and patients with acute febrile disease. The real-time RT-PCR amplified the Piry, Carajas, Alagoas and Indiana Vesiculovirus at a melting temperature 81.02 ± 0.8ºC, and the sensitivity of assay was estimated in 10 RNA copies/mL to the Piry Vesiculovirus. The viral genome has been detected in samples of horses and cattle, but not detected in human sera or arthropods. Thus, this assay allows a preliminary differential diagnosis of VSV infections.

Changing landscape of acute encephalitis syndrome in India: A systematic review.

Background. Seasonal outbreaks of acute encephalitis syndrome (AES) occur with striking regularity in India and lead to substantial mortality. Several viruses, endemic in many parts of India, account for AES. Although Japanese encephalitis virus (JEV) is a key aetiological agent for AES in India, and has attracted countrywide attention, many recent studies suggest that enteroviruses and rhabdoviruses might account for outbreaks of AES. We did a systematic review of published studies to understand the changing landscape of AES in India. Methods. Data sources: Electronic databases (PubMed, Web of Science and BIOSIS) from the start of the database to 2010. We also hand-searched journals and screened reference lists of original articles, reviews and book chapters to identify additional studies. Study selection: We included studies only on humans and from three time-periods: pre-1975, 1975–1999 and 2000– 2010. Data extraction: Independent, duplicate data extraction and quality assessment were conducted. Data extracted included study characteristics, type of study and aetiological agent identified. Data synthesis: Of the 749 unique published articles screened, 57 studies met the inclusion criteria (35 outbreak investigations and 22 surveillance studies). Results. While most studies from 1975 to 1999 identified JEV as the main cause of AES, many studies published after 2000 identified Chandipura and enteroviruses as the most common agents, in both outbreaks and surveillance studies. Overall, a positive yield with respect to identification of aetiological agents was higher in outbreak investigations as compared to surveillance studies. Conclusion. The landscape of AES in India has changed in the previous decade, and both outbreak investigations and surveillance studies have increasingly reported non-JEV aetiologies. Because of these findings, there is a need to explore additional strategies to prevent AES beyond vector control and JEV vaccination.

Viral contamination during sequential phacoemulsification surgeries in an experimental model / Contaminação viral durante cirurgias sequenciais de facoemulsificação em um modelo experimental

PURPOSE: To determine the incidence of Piry virus contamination among surgical instruments used with disposable accessories for phacoemulsification during sequential surgeries. METHODS: An experimental model was created with 4 pigs' eyes that were contaminated with Piry virus and 4 pigs' eyes that were not contaminated. Phacoemulsification was performed on the eyes, alternating between the contaminated and non-contaminated eyes. From one surgery to another, the operating fields, gloves, scalpel, tweezers, needles, syringes, tips and bag collector from the phacoemulsification machine were exchanged; only the hand piece and the irrigation and aspiration systems were maintained. RESULTS: In the collector bag, three samples from the contaminated eyes (3/4) were positive, and two samples from the non-contaminated (2/4) eyes were also positive; at the tip, one sample from the contaminated eyes (1/4) and two samples of the non-contaminated eyes (2/4) yielded positive results. In the irrigation system, one sample from a non-contaminated eye (1/4) was positive, and in the aspiration system, two samples from contaminated eyes (2/4) and two samples from non-contaminated eyes (2/4) were positive. In the gloves, the samples were positive in two samples from the non-contaminated eyes (2/4) and in two samples from the contaminated eyes (2/4). In the scalpel samples, three contaminated eyes (3/4) and none of the non-contaminated eyes (0/4) were positive; finally, two samples from the anterior chambers of the non-contaminated eyes gathered after surgery were positive. CONCLUSIONS: In two non-contaminated eyes, the presence of genetic material was detected after phacoemulsification surgery, demonstrating that the transmission of the genetic material of the Piry virus occurred at some point during the surgery on these non-contaminated eyes when the hand piece and irrigation and aspiration systems were reused between surgeries.

OBJETIVO: Determinar a incidência de contaminação com o vírus Piry entre os instrumentos cirúrgicos e acessórios usados durante cirurgias sequenciais de facoemulsificação. MÉTODOS: Um modelo experimental foi realizado com quatro olhos de porcos que foram contaminados com o vírus Piry e quatro olhos de porcos não contaminados. A facoemulsificação foi realizada alternando um olho contaminado para outro olho não contaminado. Entre as cirurgias, os campos de operação, luvas, bisturi, pinças, agulhas, seringas, pontas e bolsa coletora foram trocados, mantendo somente a caneta e os sistemas de irrigação e aspiração do facoemulsificador. RESULTADOS: No saco coletor, três amostras de olhos contaminados (3/4) foram positivos, e duas amostras de olhos não contaminados (2/4) também foram positivos; na ponta do facoemulsificador, uma amostra dos olhos contaminados (1/4) e duas amostras de olhos não contaminados (2/4) apresentaram resultados positivos. No sistema de irrigação, uma amostra de um olho não contaminado (1/4) foi positivo, e no sistema de aspiração, duas amostras de olhos contaminados (2/4) e duas amostras de olhos não contaminados (2/4) foram positivos. Nas luvas, as amostras foram positivos em dois olhos não contaminados (2/4) e duas amostras de olhos contaminados (2/4). Nas amostras de bisturi, três olhos contaminados (3/4) e nenhum dos olhos não contaminados (0/4) foram positivos e, finalmente, duas amostras da câmara anterior dos olhos não contaminados (2/4) reunidos após a cirurgia foram positivos. CONCLUSÕES: Em dois olhos não contaminados, a presença de material genético foi detectado após a cirurgia de facoemulsificação, demonstrando que a transmissão do material genético do vírus Piry ocorreu em algum ponto durante a cirurgia para estes olhos não contaminados, quando a caneta de facoemulsificação e o sistema de irrigação e aspiração foram reutilizados entre as cirurgias.

Chandipura virus encephalitis outbreak among children in Nagpur division, Maharashtra, 2007.

Background & objectives: An outbreak of acute encephalitis syndrome (AES) among children from Nagpur division, Maharashtra was investigated to confirm the aetiology and to describe clinico-epidemiological features. Methods: AES cases among children <15 yr, from Nagpur division, hospitalized between June-September 2007, were investigated. Serum and cerebrospinal fluid (CSF) were tested for IgM antibodies against Chandipura virus (CHPV) and Japanese encephalitis virus (JEV) and for CHPV RNA by RT-PCR. Partial N gene sequences were used for phylogenetic analysis. Virus isolations were attempted in rhabdomyosarcoma (RD) cell line. Sandflies were collected, pooled and tested for CHPV RNA by RT-PCR. Results: A total of 78 AES cases were recorded in children <15 yr of age. Case fatality ratio was 43.6 per cent. Male to female ratio was 1:1.2. Chandipura (CHP) was confirmed in 39 cases. CHPV RNA was detected in both CSF and serum specimens of 2 cases and in serum of 22 cases. Phylogenetic analysis showed 99.98 – 100 per cent nucleotide identity in the sequences studied. Anti-CHPV IgM antibodies were detected in CSF of 2 cases and in serum of 8 cases. Seroconversion to anti-CHPV IgM antibodies was observed in 5 cases. Clinical manifestations of CHP cases (n=38) were fever (100%), convulsion (76.3%), altered sensorium (34.2%), headache (23.7%), vomiting (44.7%) and diarrhoea (23.7%). CHPV RNA was detected in one of two pools of sandflies from affected locality. Interpretation & conclusions: Chandipura virus was confirmed as the aetiological agent of this acute encephalitis outbreak with high case-fatality among children.

Chandipura virus growth kinetics in vertebrate cell lines, insect cell lines & embryonated eggs.

Background & objectives: Since not much information on Chandipura virus is available, an attempt was made to study the growth kinetics of the virus in certain vertebrate, invertebrate cell lines and embryonated chicken eggs. Methods: Comparative study of Chandipura virus (CHPV) growth kinetics in three vertebrate cell lines [Vero E6, Rhabdo myosarcoma (RD), Porcine stable kidney (PS) cell lines], two insect cell lines [Aedes aegypti (AA) and Phlebotomus papatasi (PP-9) cell lines] and embryonated pathogen free chicken eggs was conducted, by tissue culture infective dose 50 per cent (TCID50) and indirect immunofluorescence assay (IFA). Results: All the cell lines and embryonated egg supported the growth of CHPV and yielded high virus titre. The vertebrate cell lines showed distinct cytopathic effect (CPE) within 4-6 h post infection (PI), while no CPE was observed in insect cell lines. PP-9 cell line was the most sensitive system to CHPV as viral antigen could be detected at 1 h PI by IFA. Interpretation & conclusions: Our results demonstrated that all the systems were susceptible to CHPV and achieved high yield of virus. However, the PP-9 cell line had an edge over the others due to its high sensitivity to the virus which might be useful for detection and isolation of the virus during epidemics.

Expression of porcine interferon-gamma and its safe antiviral assay / 生物工程学报

In order to ensure the biosafety of the IFN-gamma antiviral activity assay, we used a replication-deficient VSV carrying GFP as an interferon sensitive indicator virus (VSVdeltaG*G). The antiviral activities of porcine IFN-gamma expressed in Escherichia coli and in baculovirus on MDBK cells were assessed. The results showed that the antiviral activity of porcine IFN-gamma expressed in baculovirus could reach 10(5) IU/mL, while the porcine IFN-gamma expressed in E. coli showed some antiviral activity (32 IU/mL) after refolding. The results of the VSVdeltaG*G-based antiviral assay were almost identical to that of the VSV*GFP-based assay, suggesting it is highly feasible to use VSVdeltaG*G as a substitute for VSV*GFP, making assays for IFN-gamma antiviral activity safer and more accurate.

Comparison of characteristics of SVCV strains isolated in China and in Europe / 病毒学报

In the nationwide epidemiological investigation, SVCV-741 was for the first time isolated in Beijing region, China in 2003, and designated as SVCV Asian strain. In this paper, we compared SVCV-741 (Asian strains isolated in China) with SVCV-10/3 (Europe reference strain) on their physico-chemical, biological and morphological characteristics. The results indicated that there were no distinct differences between two SVCV strains on phycico-chemical and morphological characteristics. The main existing differences were: (1) The stability of SVCV-741 to temperature in cell culture was higher than that of SVCV-10/3, which might have some evolutionary and biological implication of SVCV; (2) No SVC outbreak ever occurred caused by SVCV-741;Furthermore we found that both SVCV-741 and SVCV-10/3 grew faster and produced higher virus titer in CO cells than other cell lines. It indicated that CO cell lines might be useful tool for SVCV research.

Effect of apoptosis of vesicular stomatitis virus on tumor cells of nasopharyngeal carcinoma in vivo / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To investigate apoptosis induced by vesicular stomatitis virus (VSV) in HNE-1 cancer cells of human nasopharyngeal carcinoma by models of nude mice-BALB/c in vivo.@*METHOD@#HNE-1 cells are collected from culture bottle and infected infra right-side post-back epithelium of nude mice-BALB/c (6 x 10(6) cells/0.1 ml/each mice) to create HNE-1 tumor models of nude mice-BALB/c. When the diameter of HNE-1 tumors is 5 to 8 millimeter, HNE-1 tumor models are treated with VSV (1 x 10(8) pfu /ml) or Saline. By Hoechst 33258-staining under fluorescence microscope, induction of apoptosis by VSV in HNE-1 tumor models are recorded and studied, compared with that by Saline in HNE-1 tumor models in vivo.@*RESULT@#Compared with control group of saline, apoptosis of HNE-1 cancer cells of human nasopharyngeal carcinoma increase apparently in the remaining tumor cells of nude mice treated by VSV (P < 0.01).@*CONCLUSION@#The present study suggests that the treatment with VSV could augment the apoptosis cells of human nasopharyngeal carcinoma in vivo.

Effect of apoptosis and anti-tumor of vesicular stomatitis virus on tumor cells of nasopharyngeal carcinoma in vitro / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To study the result of HNE-1 cancer cells lesion and HNE-1 cancer cells apoptosis caused by vesicular stomatitis virus in vitro.@*METHOD@#Firstly, HNE-1 cancer cells of human nasopharyngeal carcinoma were cultured in vitro. Secondly, tumor cells were treated with vesicular stomatitis virus (VSV) in different concentration (0.1 moi, 1.0 moi, 10.0 moi and 100.0 moi), while cells without treatment were used as blank assay. Finally, we observed the lesion of HNE-1 cells treated with VSV for 24 to 48 hours under invert microscope, compared with HNE-1 cells of blank assay. By measurement of MTT reduction assay and Hoechst 33258- staining under fluorescence microscope, the effect of VSV killing HNE-1 cancer cells and the induction of apoptosis by VSV in HNE-1 cancer cells were investigated in vitro.@*RESULT@#Compared with blank assay, HNE-1 cancer cells of human nasopharyngeal carcinoma treated with VSV showed phenomenon of lesion under invert microscope. Followed the increase of concentration of VSV, cell survival rate of HNE-1 cancer cells decreased. The research methods of MTT reduction assay and Hoechst 33258 staining under fluorescence microscope defined the effect of VSV killing HNE-1 cancer cells and confirmed that VSV inducing apoptosis of HNE-1 cancer cells in vitro.@*CONCLUSION@#VSV not only exhibited the anti-tumor activity in HNE-1 cancer cells of human nasopharyngeal carcinoma but also showed the potential power of inducing apoptosis of HNE-1 cancer cells in vitro.

Expression of porcine gamma-interferon in recombinant baculovirus and determination of its antiviral activity / 生物工程学报

The full-length porcine interferon gamma(PoIFN-gamma) cDNA, including the secretion signal peptide coding region was recloned into honor plasmid pFastBac 1 of Bac-To-Bac Baculovirus Expression System. These recombinant plasmids, pFastBac -PoIFN-gamma, were transformed into DH(10Bac) host bacteria to get recombinant shuttle plasmids, rBacmid-PoIFN-gamma. Recombinant baculovirus, rBac-PoIFN-gamma, was generated for expressing PoIFN-gamma, by transfecting rBacmid-PoIFN-gamma with Cellfectin Reagent into sf9 insect cells. The expression of PoIFN-gamma in insect cells was confirmed by Western Blot, indirect immunofluorescence assay and indirect ELISA. The antiviral activity assay shows that PoIFN-gamma expressed by the rBac-PoIFN-gamma can efficiently inhibit the replication of the recombinant Vesicular Stomatitis Virus expressing green fluorescence protein in PK-15 cells. The antiviral activity of PoIFN-gamma can be specifically blocked by anti-PoIFN-gamma mouse serum. The antiviral titer of culture supernatant of insect cells infected by rBac-PoIFN-gamma is 2 x 10(4) IU/mL. The results demonstrat that the rBac-PoIFN-gamma can express rPoIFN-gamma efficiently and rPoIFN-gamma has high antiviral activity.

Propagation of Chandipura virus in chick embryos.

Stocks of three Indian Chandipura virus (CHPV) isolates; one isolate from an adult febrile case in 1965 from Chandipura town, Maharashtra, and two isolates from two pediatric encephalitis cases from Andhra Pradesh, 2003 were inoculated in 10-day-old chick embryos by allantoic route. All three virus isolates replicated in chick embryos showing titre of log 10(12) to log 10(13) EID50. The results demonstrated that chick embryos are susceptible to CHPV and virus grows to high titres in this system. Therefore chick embryos can be used as an alternative host system for cultivation and isolation of CHPV as they are less expensive than laboratory animals and have several other advantages over cell cultures. Also this system can be used for the development of vaccine and diagnostic reagents.

Diagnosis of Brazilian vesiculoviruses by reverse transcription-polymerase chain reaction

We describe a reverse transcription-polymerase chain reaction (RT-PCR) and a nested-PCR for diagnosis of Piry, Carajás, Cocal, and Alagoas vesiculoviruses from Brazil. The RNA extracts of viral and clinical samples were submitted to a RT-PCR using Vesiculovirus G primers that amplify part of the glycoprotein gene. The RT-PCR produced amplicons of expected size, 290 base pair, for the four studied viruses. The RT-PCR showed a high sensitivity being 151.3 times (2.18 log) more sensitive for the detection of Piry virus than the classical procedure for virus detection in tissue culture based on the viral cytophatic effect. Amplicons had nucleotides sequenced and were aligned in order to select internal primers for a nested-PCR to confirm the origin of Piry, Carajás, Cocal, and Alagoas Vesiculovirus. Ten blood and tarsal pad epithelial samples of infected Guinea-pigs had Vesiculovirus genome amplified by RT-nested-PCR.

Virus induced chromosomal abnormalities in Chinese hamster lung cell line and human peripheral blood leukocyte culture.

Chinese hamster lung (CHL) cells were susceptible to Herpes Simplex type-1 and Chandipura viruses; which induced chromosomal abnormalities in these cells. Chromosomal changes induced in these cells were specific. The cells were refractory to measles virus and chromosomal abnormalities were not detected after inoculation of the virus. On the other hand human peripheral blood (HPB) leukocytes were susceptible to all the 3 viruses studied and exhibited chromosomal abnormalities upon infection. The aberrations induced in HPBL cultures were random. The results suggest that a virus could induce chromosomal changes only in susceptible cells. This is the first report of comparative in vitro study on chromosomes.

Epidemiology of arboviruses in Sri Lanka

This dissertation includes studies on the epidemiology of Japanese encephalitis, California serogroup viruses, Alphaviruses, Chandipura virus (Rhabdoviridae), Bhanja and Batai viruses. Seroepidemiological studies, both cross sectional and longitudinal, were carried out on human and animal sera. The serological technique used was the plaque reduction neutralization test (PRNT). Limited studies on virus isolation from field caught mosquitoes were used to supplement the seroepidemiological data. Techniques used for isolation of virus from field caught mosquitoes viz. inoculation of suckling mice, vertebrate and mosquito cell culture; and a microplate modification of PRNT were optimized. Japanese Encephalitis (JE): Serological studies on domestic animals showed that pigs were the most sensitive indicator of JE activity. Therefore, pigs were used as indicators of JE in cross sectional and longitudinal studies in different agroclimatological areas of the country. The main conclusions were that porcine JE seropositivity decreases with increasing altitude, and there is little JE activity in the wet zone highlands (\> 3000 ft) of Sri Lanka. The highest human antibody prevalence was found in the Dry Zone areas. Human seropositivity did not correlate in a linear manner with pig seropositivity. Although Randy has a significant (47percent) porcine JE infection rate, human infection is uncommon. The 'spillover' of infection to domestic animal species viz. cattle and goat decreases with increasing altitude and does correlate with porcine seropositivity. It is concluded that pig husbandry in the wet zone highlands would probably not be a human JE risk. Porcine infection in the Mahaweli C, Anuradhapura and Welisara (low elevation areas) occurred in a focused and synchronous manner. In contrast, porcine infection in Kandy was asynchronous and covered a period of six months. The period of maximal JE activity in the dry zone was the "Maha" cultivation season, whereas in the wet zone (Welisara; Kandy) it was the Yale season. JE virus was isolated from field caught Culex pseudovishnui mosquitoes in the Kandy area. California serogroup viruses: The prevalence of antibody to California serogroup viruses, viz. Lumbo (LUM). Snow Shoe Hare (SSH). and Trivittatus (TVT) were studied in man and animals in different parts of the country. Serological evidence in man and animals for a LUM related virus was obtained in many parts of the island. In addition infection of man and animals by SSH and Melao related viruses was likely. The role of these viruses in human encephalitis deserves further study. Alphaviruses: A Sagiyama related virus was isolated from Culex fuscoeephala and Culex tritaeniorhynchus mosquitoes collected in Anuradhapura. Antibody prevalence to the local isolate shows low seroprevalence in man and animal in many parts of the country, with the exception of pigs. Chikungunya and Sindbis virus activity was not detectable. Chandipura: Serological evidence for Chandipura virus infection was obtained in man and animals for the first time in Sri Lanka. The highest seroprevalence was seen in cattle. Batai and Bhanja viruses: An extension of serological studies was conducted in man and animals to Bhanja virus. Serological evidence of infection in man is recorded. A study of Batai virus was carried out on human sera and one human seroconversion associated with febrile illness was documented fer the first time with this virus. Seven arboviruses were isolated from field caught mosquitoes during the present study. In addition tc JE and Sagiyama (refer above), Arkonam virus was isolated from Sri Lanka for the first time. The mosquito vector was Cx.fuscccephala. Three other isolates remain unidentified, and have been sent to a reference laboratory for study.