Detection of bacteriophage tracers in bivalve molluscan shellfish

Procedures to enumerate commonly used bacteriophage tracers in bivalve molluscan shellfish were evaluated. Bacteriophages specific to Serratia marcescens, Escherichia coli and Enterobacter cloacae can be recovered from shellfish flesh by chloroform treatment of homogenates and subsequent clarification of samples by centrifugation. Bacteriophages were enumerated using a soft agar overlay technique. Hard clams appeared to release toxic compounds during homogenization which dramatically reduced counts of Ent. clocae bacteriophage.     

Levels of male-specific RNA bacteriophage and Escherichia coli in molluscan bivalve shellfish from commercial harvesting areas

AIMS: Current measures for controlling the public health risks associated with bivalve molluscan shellfish consumption rely on the use of Escherichia coli to indicate the sanitary quality of shellfish harvesting areas. However, it has been demonstrated that E. coli is an inadequate indicator of the viral risk associated with shellfish. An alternative indicator organism, male-specific RNA (FRNA) bacteriophage has been proposed for this role. This study compared the distribution of E. coli and FRNA bacteriophage in shellfish harvesting areas. METHODS AND RESULTS: A total of 608 shellfish samples from 49 shellfish harvesting areas were analysed for E. coli and FRNA bacteriophage using standard published methods. The geometric mean concentration of FRNA bacteriophage in all samples was over three times greater than that of E. coli (1800 and 538 counts/100 g for FRNA bacteriophage and E. coli, respectively). In contrast to E. coli, FRNA bacteriophage concentrations were strongly influenced by season with a geometric mean count of 4503 PFU/100 g in the winter (October-March) compared with 910 PFU/100 g in the summer (April-September). CONCLUSIONS: FRNA bacteriophage were present in shellfish at higher concentrations than E. coli. Elevated levels of FRNA bacteriophage observed in the winter concur with the known increased viral risk associated with shellfish harvested at that time of year in the UK. Levels of FRNA bacteriophage found in many shellfish from category B harvesting areas would not be eliminated by conventional treatment processes. SIGNIFICANCE AND IMPACT OF THE STUDY: Data from this study will inform future proposals to introduce FRNA bacteriophage as an indicator of the viral risk associated with shellfish.     

F-RNA噬菌体YM1肠道宿主分析

【背景】F-RNA噬菌体近年来常被作为水环境中诺如病毒污染的指示物。本课题组前期以大肠杆菌ATCC700891^T为宿主,从人便样中筛选出一株F-RNA噬菌体YM1,其与大肠杆菌噬菌体MS2亲缘关系最近,MS2宿主通常为含有性菌毛的雄性大肠杆菌。【目的】探索F-RNA噬菌体与其肠道宿主及诺如病毒之间的互作关系,筛选YM1的肠道宿主。【方法】采用选择性培养基筛选YM1阳性便样中的大肠杆菌并进行YM1侵染验证,结合16S rRNA基因扩增子测序分析YM1接种前后便样中的差异性菌群种类,对YM1阳性便样中潜在的YM1肠道宿主进行分析。【结果】筛选到351个大肠杆菌菌株,YM1侵染结果表明这些大肠杆菌均不是YM1的宿主;16S rRNA基因扩增子测序分析差异性菌种显示,Enterobacter sp. (OTU144)和Enterobacter sp. (OTU11)这2株肠杆菌属细菌的相对丰度在YM1感染后发生显著性的降低,表明该2种细菌可能为YM1的潜在肠道宿主。【结论】YM1具有严格的宿主特异性,便样中大肠杆菌并非YM1的肠道宿主,同时发现了2种YM1的潜在宿主,为进一步筛选分离YM1的肠道宿主提供了方向和依据。     

F-specific RNA bacteriophages are adequate model organisms for enteric viruses in fresh water.

Culturable enteroviruses were detected by applying concentration techniques and by inoculating the concentrates on the BGM cell line. Samples were obtained from a wide variety of environments, including raw sewage, secondary effluent, coagulated effluent, chlorinated and UV-irradiated effluents, river water, coagulated river water, and lake water. The virus concentrations varied widely between 0.001 and 570/liter. The same cell line also supported growth of reoviruses, which were abundant in winter (up to 95% of the viruses detected) and scarce in summer (less than 15%). The concentrations of three groups of model organisms in relation to virus concentrations were also studied. The concentrations of bacteria (thermotolerant coliforms and fecal streptococci) were significantly correlated with virus concentrations in river water and coagulated secondary effluent, but were relatively low in disinfected effluents and relatively high in surface water open to nonhuman fecal pollution. The concentrations of F-specific RNA bacteriophages (FRNA phages) were highly correlated with virus concentrations in all environments studied except raw and biologically treated sewage. Numerical relationships were consistent over the whole range of environments; the regression equations for FRNA phages on viruses in river water and lake water were statistically equivalent. These relationships support the possibility that enteric virus concentrations can be predicted from FRNA phage data.     

A molecular method for the recovery and identification of enteric virus in shellfish

In this paper we report the results of an investigation into the presence of enteric viruses in shellfish from the waters around Sardinia. Twenty two samples of shellfish were examined using a rapid and sensitive technique to concentrate and detect viral RNA in shellfish tissues. After recovery of viral particles, RNA was extracted, transcribed into cDNA and amplified using "nested PCR". Testing with enterovirus-specific RT-PCR produced positive results in over 13% of specimens. The virus detection procedure appears to be effective. In some circumstances it could be a better test of water quality than conventional monitoring techniques.     

A tentative national reference procedure for isolation and enumeration of Escherichia coli from bivalve molluscan shellfish by most probable number method

In the UK several quantitative methods exist for the examination of bivalve molluscan shellfish for sewage contamination. These methods include roll tubes, pour plates and most probable number (MPN) techniques, but there is no national standard method. A comparative study was made of the most commonly used methods for detection of Escherichia coli in bivalve shellfish. Schemes employing solid media, such as the roll tube and pour plate methods, underestimated faecal contamination in shellfish tissue compared with a liquid MPN multiple test-tube method using minerals-modified-glutamate broth (MMGB) as primary enrichment medium. The composition of MMGB apparently permits repair of sublethally injured cells of E. coli. Incorporation of resuscitation stages into the pour plate technique did not yield higher counts. A standardized MPN technique for examination of bivalve molluscan shellfish for E. coli content is proposed as a possible national reference procedure pending further collaborative assessment.     

A tentative national reference procedure for isolation and enumeration of Escherichia coli from bivalve molluscan shellfish by most probable number method

In the UK several quantitative methods exist for the examination of bivalve molluscan shellfish for sewage contamination. These methods include roll tubes, pour plates and most probable number (MPN) techniques, but there is no national standard method. A comparative study was made of the most commonly used methods for detection of Escherichia coli in bivalve shellfish. Schemes employing solid media, such as the roll tube and pour plate methods, underestimated faecal contamination in shellfish tissue compared with a liquid MPN multiple test-tube method using minerals-modified-glutamate broth (MMGB) as primary enrichment medium. The composition of MMGB apparently permits repair of sublethally injured cells of E. coli. Incorporation of resuscitation stages into the pour plate technique did not yield higher counts. A standardized MPN technique for examination of bivalve molluscan shellfish for E. coli content is proposed as a possible national reference procedure pending further collaborative assessment.     

A candidate gene for human U1 RNA

[This corrects the article on p. 133 in vol. 1, PMID: 6201353.].     

Theiler's murine encephalomyelitis virus as a vaccine candidate for immunotherapy

The induction of sterilizing T-cell responses to tumors is a major goal in the development of T-cell vaccines for treating cancer. Although specific components of anti-viral CD8+ immunity are well characterized, we still lack the ability to mimic viral CD8+ T-cell responses in therapeutic settings for treating cancers. Infection with the picornavirus Theiler's murine encephalomyelitis virus (TMEV) induces a strong sterilizing CD8+ T-cell response. In the absence of sterilizing immunity, the virus causes a persistent infection. We capitalized on the ability of TMEV to induce strong cellular immunity even under conditions of immune deficiency by modifying the virus to evaluate its potential as a T-cell vaccine. The introduction of defined CD8+ T-cell epitopes into the leader sequence of the TMEV genome generates an attenuated vaccine strain that can efficiently drive CD8+ T-cell responses to the targeted antigen. This virus activates T-cells in a manner that is capable of inducing targeted tissue damage and glucose dysregulation in an adoptive T-cell transfer model of diabetes mellitus. As a therapeutic vaccine for the treatment of established melanoma, epitope-modified TMEV can induce strong cytotoxic T-cell responses and promote infiltration of the T-cells into established tumors, ultimately leading to a delay in tumor growth and improved survival of vaccinated animals. We propose that epitope-modified TMEV is an excellent candidate for further development as a human T-cell vaccine for use in immunotherapy.     

A human multi-epitope recombinant vaccinia virus as a universal T cell vaccine candidate against influenza virus

There is a need to develop a universal vaccine against influenza virus infection to avoid developing new formulations of a seasonal vaccine each year. Many of the vaccine strategies for a universal vaccine target strain-conserved influenza virus proteins, such as the matrix, polymerase, and nucleoproteins, rather than the surface hemagglutinin and neuraminidase proteins. In addition, non-disease-causing viral vectors are a popular choice as a delivery system for the influenza virus antigens. As a proof-of-concept, we have designed a novel influenza virus immunogen based on the NP backbone containing human T cell epitopes for M1, NS1, NP, PB1 and PA proteins (referred as NPmix) as well as a construct containing the conserved regions of influenza virus neuraminidase (N-terminal) and hemagglutinin (C-terminal) (referred as NA-HA). DNA vectors and vaccinia virus recombinants expressing NPmix (WR-NP) or both NPmix plus NA-HA (WR-flu) in the cytosol were tested in a heterologous DNA-prime/vaccinia virus-boost vaccine regimen in mice. We observed an increase in the number of influenza virus-specific IFNγ-secreting splenocytes, composed of populations marked by CD4(+) and CD8(+) T cells producing IFNγ or TNFα. Upon challenge with influenza virus, the vaccinated mice exhibited decreased viral load in the lungs and a delay in mortality. These findings suggest that DNA prime/poxvirus boost with human multi-epitope recombinant influenza virus proteins is a valid approach for a general T-cell vaccine to protect against influenza virus infection.     

A candidate gene for human U1 RNA.

Clones containing sequences complementary to the small nuclear RNA U1 were isolated from the human DNA library of Lawn et al. (1978). Three clones were studied by hybridization and restriction enzyme cleavage. The results showed that the inserts in all three clones were different and that each clone contains one single copy of a sequence which hybridizes to U1 RNA. The results revealed moreover that only one of the three clones contains all the cleavage sites which can be predicted from the known sequence of human U1 RNA, suggesting that the three clones comprise one candidate U1 gene and two pseudogenes. A fragment from the recombinant with the candidate U1 gene was subcloned in the pPR322 plasmid and part of its sequence was determined. The results showed that the subclone contains a sequence which matches that of the human U1 RNA perfectly. The sequence "TATAT" which often is found adjacent to RNA polymerase II start sites, was identified 33-37 base pairs upstream from the beginning of the U1 sequence. Two ten base pairs long, nearly perfect, direct repeats were also identified in the vicinity of the U1 sequence and an imperfect inverted repeat follows immediately after the U1 gene.     

我国沿海主要经济贝类中典型人类肠道病毒的污染分布

【目的】了解我国沿海主要经济贝类中肠道病毒的污染分布对保障贝类食用安全具有重要作用。【方法】通过建立5种典型人类肠道病毒的特异、灵敏、高通量的基因芯片检测技术,开展全国范围内沿海主要海水增养殖区经济贝类体内肠道病毒的污染调查。【结果】采集的162份经济贝类中肠道病毒的阳性检出率分别为:甲肝病毒4.3%;诺如病毒14.8%;轮状病毒6.2%;星状病毒5.6%;腺病毒9.9%,其中诺如病毒污染居于首位,该检测结果与PCR结果高度一致。我国沿海省市主要养殖区内贝类均受到了肠道病毒不同程度的污染,且不同省市之间差异不显著。在选取的6类经济贝类中,牡蛎中肠道病毒阳性检出率最高,其次是毛蚶和泥蚶。【结论】本研究表明,在我国,沿海主要经济贝类受到肠道病毒污染是一种较普遍的现象,给人类健康带来了潜在的危害,本研究为贝类食品的安全监控提供了基础数据资料。     

Comparison of bacteriophage and enteric virus removal in pilot scale activated sludge plants

AIMS: The aim of this experimental study was to determine comparatively the removal of two types of bacteriophages, a somatic coliphage and an F-specific RNA phage and of three types of enteric viruses, hepatitis A virus (HAV), poliovirus and rotavirus during sewage treatment by activated sludge using laboratory pilot plants. METHODS AND RESULTS: The cultivable simian rotavirus SA11, the HAV HM 175/18f cytopathic strain and poliovirus were quantified by cell culture. The bacteriophages were quantified by plaque formation on the host bacterium in agar medium. In each experiment, two pilots simulating full-scale activated sludge plants were inoculated with viruses at known concentrations, and mixed liquor and effluent samples were analysed regularly. In the mixed liquor, liquid and solid fractions were analysed separately. The viral behaviour in both the liquid and solid phases was similar between pilots of each experiment. Viral concentrations decreased rapidly following viral injection in the pilots. Ten minutes after the injections, viral concentrations in the liquid phase had decreased from 1.0 +/- 0.4 log to 2.2 +/- 0.3 log. Poliovirus and HAV were predominantly adsorbed on the solid matters of the mixed liquor while rotavirus was not detectable in the solid phase. In our model, the estimated mean log viral reductions after 3-day experiment were 9.2 +/- 0.4 for rotavirus, 6.6 +/- 2.4 for poliovirus, 5.9 +/- 3.5 for HAV, 3.2 +/- 1.2 for MS2 and 2.3 +/- 0.5 for PhiX174. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that the pilots are useful models to assess the removal of infectious enteric viruses and bacteriophages by activated sludge treatment. Our results show the efficacy of the activated sludge treatment on the five viruses and suggest that coliphages could be an acceptable indicator of viral removal in this treatment system.     

Hexamer of bacteriophage f2 coat protein as a repressor of bacteriophage RNA polymerase synthesis.

Formation of complex I between phage f2 RNA and coat protein, leading to repression of phage RNA polymerase synthesis, depends nonlinearly upon the concentration of the coat protein. Maximum formation of complex I was observed when six molecules of coat protein were bound to one molecule of RNA. RNase digestion of a glutaraldehyde-fixed complex left, as the products, coat protein oligomers. The heaviest, hexamers, predominated in the mixture. It was also shown that, in an ionic environment required for phage protein synthesis, coat protein at a concentration optimum for complex I formation exists in solution as a dimer. The results indicate that the translational repression of the RNA polymerase cistron is due to a cooperative attachment to phage template of three dimers of coat protein, forming a hexameric cluster on an RNA strand.     

Evaluation of two human plasma pools as candidate international standard preparations for syphilitic antibodies

A collaborative study was designed to asses two freeze-dried human plasma preparations containing anti-Treponema pallidum antibodies, 05/132 and 05/122, for their suitability as international reference reagents for syphilis serology. Both preparations are intended as replacements of the first international standard (IS) for syphilitic serum antibodies (HS). Samples were tested by eight laboratories using the T. pallidum passive particle agglutination assay (TPPA), the venereal disease research laboratory test (VDRL) and the rapid plasma reagin test (RPR). In addition a range of immunoassays was also used. The outcome of the collaborative study revealed that candidate standard 05/132 contains T. pallidum-specific IgG and IgM and is reactive in VDRL or RPR, and that 05/122 contains T. pallidum-specific IgG but is not reactive in either the VDRL or RPR test. Both 05/132 and 05/122 are reactive in the TPPA. On the basis of these results the Expert Committee on Biological Standardization of the World Health Organization designated 05/132 as the 1st IS for human syphilitic plasma IgG and IgM with a unitage of 3 IU per ampoule relative to HS and 05/122 as the 1st IS for human syphilitic plasma IgG with a unitage of 300 mIU per ampoule relative to 05/132.     

Leptospiral glycolipoprotein as a candidate antigen for serodiagnosis of human leptospirosis

Aims:  Development of a simple, specific, rapid and inexpensive Dot-ELISA test for early diagnosis of human leptospirosis.
Methods and Results:  Serum samples from 90 patients diagnosed with leptospirosis were analysed by Dot-ELISA test incorporating Glycolipoprotein (GLP) antigen from serovars Copenhageni and Patoc. Results were compared with those obtained with microscopic agglutination test, currently, the gold standard reference serological method. Serum samples from healthy blood bank donors and patients diagnosed with diseases other than leptospirosis were used as negative controls. The specificities of both GLP-based assays were 97·1% and 100% with serum samples from patients with other diseases and with serum samples from healthy control group, respectively. With serum samples from patients with acute leptospirosis, sensitivity was 76·6% with Dot-ELISA Copenhageni and 90·0% with Dot-ELISA Patoc. With serum samples from patients in convalescence, sensitivity was 100% with both GLP-based assays.
Conclusions:  This Dot-ELISA provides a candidate antigen for serodiagnosis of leptospirosis during all phases of illness and could be a good alternative method for the early diagnosis of leptospirosis.
Significance and Impact of the Study:  The Dot-ELISA test is simple, specific, rapid and inexpensive. It is suitable for identifying a large number of samples and, hence, reducing the death rate of patients with leptospirosis.