Epidemiology and Phylogenetic Analysis of Human Rhinovirus/Enterovirus in Odisha,Eastern India

Introduction: Human rhinovirus (HRV) and Enterovirus (ENV) are the major causes of childhood acute respiratory tract infections (ARTIs). This study sought to understand the distribution pattern of HRV subgroups, their seasonality and association with respiratory complications in patients at a tertiary care hospital. Results: Of the total 332 ARTI samples, 82 (24.7%) were positive for ENV/HRV. Twenty positive samples were processed further for phylogenetic analysis. Ten of the 20 samples were identified to be HRVs (70% HRV A and 30% HRV C) and nine were enteroviruses. HRV A clustered near three distinct HRV types (A12, A78 and A82). Four of the HRV strains (represented as SEQ 137 rhino, SEQ 282 rhino, SEQ 120 rhino and SEQ 82 rhino) had high sequence similarity. HRV C showed seasonality and was associated with disease severity. Conclusion: The genotyping and phylogenetic analysis of the HRVs in the current study shows its circulatory pattern, association with risk factors and evolutionary dynamics.     

Trophic action of pharmacological substances with a guanidine group on mouse neuroblastoma cells and chick ganglionic neurons in culture

A series of substances (designated CTQ compounds) with a guanidine group have been synthesized and tested for their ability to promote neuronal survival and neurite outgrowth. Mouse neuroblastoma clonal cell lines grown in serum-containing medium for 10 days as well as primary cultures of embryonic chicken ganglion neurons grown in serum-free defined medium for 1 or 2 days have been used for the experiments. Among the various CTQ compounds (CTQ1–CTQ20) tested, only CTQ8 exerted positive neurotrophic effects on these peripheral neuronal cells. At a concentration of 10⁻⁴ M, CTQ8 enhanced neuritogenesis of neuroblastoma cells. However, the most striking influence of CTQ8 was its promoting effect (6- to 10-fold) on the survival of chicken ciliary and dorsal root ganglionic neurons at concentrations ranging from 10⁻³ M to 5×10⁻⁴ M.     

Respiratory virus induction of alpha-, beta- and lambda-interferons in bronchial epithelial cells and peripheral blood mononuclear cells

Background: Respiratory viruses, predominantly rhinoviruses are the major cause of asthma exacerbations. Impaired production of interferon‐β in rhinovirus infected bronchial epithelial cells (BECs) and of the newly discovered interferon‐λs in both BECs and bronchoalveolar lavage cells, is implicated in asthma exacerbation pathogenesis. Thus replacement of deficient interferon is a candidate new therapy for asthma exacerbations. Rhinoviruses and other respiratory viruses infect both BECs and macrophages, but their relative capacities for α‐, β‐ and λ‐interferon production are unknown. Methods: To provide guidance regarding which interferon type is the best candidate for development for treatment/prevention of asthma exacerbations we investigated respiratory virus induction of α‐, β‐ and λ‐interferons in BECs and peripheral blood mononuclear cells (PBMCs) by reverse transferase‐polymerase chain reaction and enzyme‐linked immunosorbent assay. Results: Rhinovirus infection of BEAS‐2B BECs induced interferon‐α mRNA expression transiently at 8 h and interferon‐β later at 24 h while induction of interferon‐λ was strongly induced at both time points. At 24 h, interferon‐α protein was not detected, interferon‐β was weakly induced while interferon‐λ was strongly induced. Similar patterns of mRNA induction were observed in primary BECs, in response to both rhinovirus and influenza A virus infection, though protein levels were below assay detection limits. In PBMCs interferon‐α, interferon‐β and interferon‐λ mRNAs were all strongly induced by rhinovirus at both 8 and 24 h and proteins were induced: interferon‐α>‐β>‐λ. Thus respiratory viruses induced expression of α‐, β‐ and λ‐interferons in BECs and PBMCs. In PBMCs interferon‐α>‐β>‐λ while in BECs, interferon‐λ>‐β>‐α. Conclusions: We conclude that interferon‐λs are likely the principal interferons produced during innate responses to respiratory viruses in BECs and interferon‐αs in PBMCs, while interferon‐β is produced by both cell types.     

Antagonism of the ruthenium red-induced paralysis in mice by 4-aminopyridine, guanidine and lanthanum

4-Aminopyridine and guanidine were administered intraperitoneally to mice during the complete flaccid paralysis induced by treatment with ruthenium red (RuR). At 1-8 min after 4-aminopyridine or guanidine the animals had recovered completely from the paralysis, whereas the control mice injected only with RuR remained paralytic for at least 60 min. Intraperitoneal injections of LaCl3 had no apparent effects on animal motility and did not reverse the paralysis produced by RuR. However, when La3 + was administered 30 min prior to RuR the occurrence of flaccid paralysis was totally prevented. The results obtained are discussed in terms of the possible antagonist effects of the compounds used on acetylcholine release at neuromuscular junctions.     

Mechanisms of HIV persistence in HIV reservoirs

The establishment and maintenance of HIV reservoirs that lead to persistent viremia in patients on antiretroviral drugs remains the greatest challenge of the highly active antiretroviral therapy era. Cellular reservoirs include resting memory CD4+ T lymphocytes, implicated as the major HIV reservoir, having a half‐life of approximately 44 months while this is less than 6 hours for HIV in plasma. In some individuals, persistent viremia consists of invariant HIV clones not detected in circulating resting CD4+ T lymphocytes suggesting other possible sources of residual viremia. Some anatomical reservoirs that may harbor such cells include the brain and the central nervous system, the gastrointestinal tract and the gut‐associated lymphoid tissue and other lymphoid organs, and the genital tract. The presence of immune cells and other HIV susceptible cells, occurring in differing compositions in anatomical reservoirs, coupled with variable and poor drug penetration that results in suboptimal drug concentrations in some sites, are all likely factors that fuel the continued low‐level replication and persistent viremia during treatment. Latently, HIV‐infected CD4+ T cells harboring replication‐competent virus, HIV cell‐to‐cell spread, and HIV‐infected T cell homeostatic proliferation due to chronic immune activation represent further drivers of this persistent HIV viremia during highly active antiretroviral therapy.     

Picorna- and togavirus infection of cells detected by gas chromatography.

Viral infection of cells causes chemical and metabolic changes, which can be detected by gas chromatography (GC) of ether extracts of supernatant fluids and cell homogenates before any significant damage to the cells is observable microscopically. The characteristic and specific GC patterns obtained from BHK-21 and Vero cell cultures infected with encephalomyocarditis, polio, echoviruses, and a togavirus make it possible to distinguish between these infecting viruses. The appearance of 1 or 2 compounds, represented by GC peaks with TR values of 302 and 677 seconds seems to be specific for these viruses. Other peaks found in the supernatant media 1-2 hours after infection probably represent cell constituents leaking into the medium as a result of damage to the cell membrane by the invading virus.     

Improved detection of rhinoviruses in nasal and throat swabs by seminested RT-PCR

A seminested RT-PCR (nRT-PCR) was used to detect picornavirus (PV) RNA in cell cultures inoculated with rhinoviruses (HRVs) and enteroviruses (EVs). PCR tests in which a primary "touchdown" PCR was followed by secondary reactions using PV or HRV specific primers were able to differentiate HRVs of 48 serotypes from EVs. PVnRT-PCR and HRVnRT-PCR were then used to test nasal and throat swabs from adult subjects with naturally acquired respiratory virus infections. The swabs were also analysed for respiratory viruses by cell culture techniques and the rates of PV identification by the two methods were compared. PVnRT-PCR was found to be at least five times more sensitive than cell culture for the detection of PVs in these clinical specimens. Paired acute and convalescent serum samples were tested for complement fixing antibodies to adenovirus, influenza A and B, respiratory syncytial virus, parainfluenza viruses 1, 2, and 3, Myco plasma pneumoniae, and Chlamydia psittaci. An enzyme-linked immunosorbent assay (ELISA) was used to detect rises in antibody level to coronavirus types 229E and OC43. The overall rate of pathogen identification in 159 swabs from adult asthmatics increased from 28% when only cell culture and serology were used to 57% when these methods were supplemented by PVnRT-PCR. © 1993 Wiley-Liss, Inc.     

反义寡聚核苷酸对CVB3蛋白基因表达的抑制作用及量效关系的研究

目的 观察病毒基因组5′端非编码区内与翻译起始有关的位点和结构蛋白编码区的特异性反义核酸对病毒蛋白表达的影响及其量效关系。方法 应用病毒致细胞病变作用保护实验、空斑形成实验和空斑形成减少实验、Western blot实验等方法，观察特定的反义核酸抑制病毒感染的效果。结果 针对IRES位点、AUG区域和VPl区的3条反义核酸Scb561、Scb733、Scb2785，对病毒感染有明显的抑制作用。病毒的感染量为0．01MOI时，3种反义核酸在5Fmol／L时对病毒的抑制率均在90％以上，当病毒增加到10MOI时，抑制率仍在50％以上。Scb561和Scb733可明显的抑制病毒基因表达，当Scb561的浓度在0．625μmol／L时感染后的细胞内病毒蛋白量明显减少，增加到2．5／lmol／L时病毒蛋白表达几乎看不到。另外Scb561、Scb733剂量与抗病毒效果呈现正相关关系。随着Scb561和Scb733浓度的增加，其抗病毒活性也随之增加直到抑制率达到90％以上，有效剂量在0．6～5μmol／L之间，且对细胞无毒性作用。非特异寡聚核苷酸对照实验显示，5μmol／L浓度时对病毒感染无明显抑制作用。结论 针对核糖体进入位点和翻译起始位点的反义核酸，有明显的特异性抑制病毒基因表达的作用。为反义寡聚核苷酸成为一个潜在的治疗病毒感染的药物提供了重要的研究基础。     

Invasion of HeLa 229 cells by virulent Bordetella pertussis.

Phase-dependent invasive behavior of Bordetella pertussis was demonstrated by recovery of viable organisms from gentamicin-treated HeLa cell monolayers and by transmission electron microscopy. Several mutants of B. pertussis with Tn5 or Tn5 lac inserted into various vir-regulated genes were evaluated for differences in their invasive abilities. Mutants lacking filamentous hemagglutinin, pertussis toxin, and two as yet uncharacterized vir-regulated products had levels of invasion significantly lower than that of the parent strain BP338. In contrast, invasion by mutants lacking adenylate cyclase toxin was significantly increased compared with that of wild-type B. pertussis. This increase in invasion was eliminated when concentrations of intracellular cyclic 3'-5' AMP were stimulated by treating HeLa cells with cholera toxin or forskolin. Entry of B. pertussis occurred through a microfilament-dependent phagocytic process, as evidenced by the marked reduction in uptake following treatment of HeLa cells with cytochalasin D. Invasion was inhibited with polyclonal anti-B. pertussis and anti-filamentous hemagglutinin antisera. In addition, a monoclonal antibody against lipooligosaccharide A reduced uptake by 65.5%. The preservation of HeLa cell integrity and the limited replication of intracellular bacteria suggest that invasion may represent a means by which B. pertussis evades an active host immune response.     

Host-range mutants of adenovirus type 5 defective for growth in HeLa cells

Host-range mutants of adenovirus type 5 have been selected on human embryo kidney cells transformed by sheared adeno 5 DNA (293 cells). These mutants grow well on 293 cells, but are restricted on HeLa cells. By complementation tests on HeLa cells, these mutants were classified into two groups, neither of which was found to correspond to any of the 17 temperature-sensitive complementation groups. Members of the two complementation groups of host-range mutants differ in their host range on other cell types: Members of one group grow only on 293 cells, while those of the other group on both normal and transformed human embryo kidney cells. In the second group, cell-associated adeno gene functions are not needed for mutant growth, but, with the other group, it is possible that an adeno gene product made in the transformed cell complements the growth of the mutants. Recombination tests carried out with the host-range mutants and Ad5 temperature-sensitive mutants indicate that the host-range mutations map within the left half of the genetic map and that some (hr 1 and hr 2) probably lie very close to the left end of the map. The results presented in this communication are briefly discussed in relation to recent unpublished work concerning the biochemical characteristics of the mutants and the transformed 293 cells and to the role of adeno genes in transformation.     

Early events in the infection process of adenovirus type 5 in HeLa cells

The fate of adenovirus type 5r after infection of HeLa cells has been studied with the help of purified labeled virus preparations. The infection process starts with the adsorption of virions on the cell surface followed by penetration into the cells. Shortly after penetration, the particles inside the cells have a similar appearance as intact virions. However, a small amount of protein must have been lost, as concluded from radioactivity measurements and from the change in buoyant density (1.35 g/cm(3) instead of 1.34 g/cm(3), the value for intact virions). Evidence is presented that antigen B is absent from the 1.35-particles. After the transformation of virions into 1.35-particles, further uncoating leads to the formation of DNase-sensitive DNA-protein complexes. The proteins released during the uncoating show a buoyant density of 1.31 g/cm(3) and are almost completely insoluble in acid. No extensive breakdown of protein can be detected. The processes of attachment, penetration, and uncoating are not inhibited by prevention of de novo protein synthesis, as could be shown with cycloheximide.     

Internalization of human rhinovirus 14 into HeLa and ICAM-1-transfected BHK cells

Virus adsorption and uptake of human rhinovirus 14 (HRV14) were studied with HeLa cells and baby hamster kidney (BHK) cells which were transfected with the HRV14 receptor intercellular adhesion molecule-1 (ICAM-1). Transmission electron microscopy of HeLa cells revealed that HRV14 was internalized via clathrin-coated pits and -coated vesicles. A minority of virus particles also used uncoated vesicles for entry. The internalization showed the characteristics of receptor-mediated endocytosis. Presence of the carboxylic ionophore monensin inhibited viral uncoating, indicating a pH-dependent entry mechanism. The expression of ICAM-1 on the surface of the ICAM-1 transfected baby hamster kidney cells (BHK-ICAM cells) allowed extensive virus adsorption and internalization through membrane channels. Virus particles were lined up in these channels like pearls on a string, but did not induce a productive infection. Although ICAM-1 was expressed to the same degree on BHK-ICAM and HeLa cells, HRV14 induced neither viral protein and RNA syntheses nor infectious virus progeny in BHK-ICAM cells. ICAM-1 on the transfected BHK cells was a functional active receptor as it rendered these cells permissive to coxsackievirus A21. These results suggest that HRV14 uptake into BHK-ICAM cells is blocked directly in or shortly after its final step of internalization, the uncoating. Our findings underline that the receptor ICAM-1 determines virus uptake into cells, however, is not sufficient to confer susceptibility of BHK cells to HRV14 infection. Received: 11 October 1996     

Coxsackievirus B3 replication and persistence in intestinal cells from mice infected orally and in the human CaCo-2 cell line

Although the transmission of coxsackievirus B3 occurs mainly via the oral route, little is known about the primary replication and persistence of this agent in the intestine. To address this question, BALB/c mice were inoculated by gavage with coxsackievirus B3, Nancy strain. The mice were killed from 1 hr to 90 days after infection. The viral markers were detected in the small intestine using RT-PCR, cell culture and detection of VP1 protein. Coxsackievirus B3 was detected positive by the three methods from hr 2 to day 45 after infection. By using monoclonal antibodies directed towards VP1, CD40 and CD26, the virus was shown to be present in the lymphocytes of the mucosa as soon as 2 hr after infection; in contrast, no virus was detected in the epithelial cells lining the intestinal lumen. Further experiments were performed to evaluate the capacity of coxsackievirus B3 to establish a persistent infection in two intestinal cell lines. In contrast to HT29 cells, the CaCo-2 cells were shown to develop a persistent infection for up to 20 passages, as demonstrated by the detection of viral RNA and VP1 protein. This study provides further evidence that, after infection by the oral route, the viral particles are concentrated in the lymphocytes of the mucosal layer. In addition, the results suggest that coxsackievirus B3 is capable of establishing a persistent infection in the small intestine that may act as a reservoir of viral particles for the delayed spread of the virus to other target organs.     

Biochemical and immunological characterization of deoxythymidine kinase of thymidine kinaseless HeLa cells biochemically transformed by herpes simplex virus type.

Thymidine kinase (TK) from herpes simplex virus type 1 (HSV-1) biochemically transformed HeLa cells, purified by affinity chromatography, has been characterized with respect to its electrophoretic mobility, molecular weight, activation energy, substrate specificity, and immunological specificity. TK purified from HSV-1-transformed HeLa cells has the same electrophoretic mobility as TK purified from HeLa cells lytically infected with HSV-1. The sedimentation velocity of purified TK from transformed cells was similar to that previously reported for the lytic enzyme, and its molecular weight was estimated to be 70,000. The activation energy of purified transformed-cell TK was 18.3 kcal/mol. Antiserum prepared against purified HSV-1 TK, although it showed some cross-reactivity, preferentially neutralized homologous TK. The transformed-cell TK antiserum also neutralized the deoxycytidine kinase activity of HSV-1-infected cell extracts but had no effect on deoxycytidine kinase activity of HSV-2-infected cell extract. These results further support the notion that TK acquired by HeLa cells transformed by HSV-1 is of viral and not of cellular origin.     

The impact of high risk human papillomavirus testing in an inner London colposcopy clinic

This is an audit of a new technique to improve the colposcopy service. Samples were tested for high risk HPV DNA using Digene Hybrid Capture II. Sixty-four percent of the sampled women under 30 had detectable high risk HPV DNA, decreasing to 44% in 30--39 year olds and to 27% in women over 40. High risk HPV prevalence increased with severity of cytology, although 22% with normal colposcopy had detectable high risk HPV. Of those women treated for cervical dysplasia, 83% had detectable high risk HPV prior to treatment, compared to only 32% afterwards. The audit has shown that high risk HPV testing has considerable discriminatory value. It has been integrated successfully into the service, particularly to manage low grade cervical abnormalities and to add valuable information following treatment for cervical dysplasia. Results need to be interpreted alongside colposcopy, cytology, and histology, and care must be taken in the interpretation of a single high risk HPV result.     

Rhinovirus induces natural killer-like cytotoxic cells and interferon alpha in mononuclear leukocytes.

Natural killer-like cellular cytotoxicity was augmented by incubation of human rhinovirus serotype 2 with peripheral blood mononuclear leukocytes collected from healthy donors. The production of alpha interferon but not gamma interferon was identified in the same cell cultures. A specific interaction of conformationally intact rhinovirus with peripheral blood mononuclear leukocytes was required for induction of the response, since the response was extinguished at reduced quantities of infectious rhinovirus, and acid inactivated rhinovirus did not augment cellular cytotoxicity. Productive replication of rhinovirus was not observed in cultures of peripheral blood mononuclear leukocytes. The replicative failure was not related merely to interferon production, since the rate of disappearance of rhinovirus was similar to that observed in cell free medium. The findings suggest that natural killer cells should be considered as a potential component of the local nasopharyngeal pathophysiology of rhinovirus infection.     

Origin of human mast cells studied by dual immunofluorescence.

Previous reports have suggested that mast cells derive either directly from basophils or mononuclear phagocytes, or from a common myeloid precursor. We have used monoclonal antibodies to investigate expression by normal human tissue mast cells of antigens characteristic of other haemopoietic lineages. We find that mast cells express panhaemopoietic markers, but not antigens typical of either myeloid or lymphoid cells. We propose, therefore, that mast cells form a distinct lineage, only distantly-related to other haemopoietic cells.