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Background

In a globalized word, prevention of infectious diseases is a major challenge. Rapid detection of viable virus particles in water and other environmental samples is essential to public health risk assessment, homeland security and environmental protection. Current virus detection methods, especially assessing viral infectivity, are complex and time-consuming, making point-of-care detection a challenge. Faster, more sensitive, highly specific methods are needed to quantify potentially hazardous viral pathogens and to determine if suspected materials contain viable viral particles. Fourier transform infrared (FTIR) spectroscopy combined with cellular-based sensing, may offer a precise way to detect specific viruses. This approach utilizes infrared light to monitor changes in molecular components of cells by tracking changes in absorbance patterns produced following virus infection. In this work poliovirus (PV1) was used to evaluate the utility of FTIR spectroscopy with cell culture for rapid detection of infective virus particles.

Results

Buffalo green monkey kidney (BGMK) cells infected with different virus titers were studied at 1 - 12 hours post-infection (h.p.i.). A partial least squares (PLS) regression method was used to analyze and model cellular responses to different infection titers and times post-infection. The model performs best at 8 h.p.i., resulting in an estimated root mean square error of cross validation (RMSECV) of 17 plaque forming units (PFU)/ml when using low titers of infection of 10 and 100 PFU/ml. Higher titers, from 103 to 106 PFU/ml, could also be reliably detected.

Conclusions

This approach to poliovirus detection and quantification using FTIR spectroscopy and cell culture could potentially be extended to compare biochemical cell responses to infection with different viruses. This virus detection method could feasibly be adapted to an automated scheme for use in areas such as water safety monitoring and medical diagnostics.  相似文献   

3.
Bunyamwera virus replication was examined in Aedes albopictus (mosquito) cell cultures in which a persistent infection is established and in cytopathically infected BHK cells. During primary infection of A. albopictus cells, Bunyamwera virus reached relatively high titers (107 PFU/ml), and autointerference was not observed. Three virus-specific RNAs (L, M, and S) and two virion proteins (N and G1) were detected in infected cells. Maximum rates of viral RNA synthesis and viral protein synthesis were extremely low, corresponding to <2% of the synthetic capacities of uninfected control cells. Viral protein synthesis was maximal at 12 h postinfection and was shut down to barely detectable levels at 24 h postinfection. Virus-specific RNA and nucleocapsid syntheses showed similar patterns of change, but later in infection. The proportions of cells able to release a single PFU at 3, 6, and 54 days postinfection were 100, 50, and 1.5%, respectively. Titers fell to 103 to 105 PFU/ml in carrier cultures. Persistently infected cultures were resistant to superinfection with homologous virus but not with heterologous virus. No changes in host cell protein synthesis or other cytopathic effects were observed at any stage of infection. Small-plaque variants of Bunyamwera virus appeared at approximately 7 days postinfection and increased gradually until they were 75 to 95% of the total infectious virus at 66 days postinfection. Temperature-sensitive mutants appeared between 23 and 49 days postinfection. No antiviral activity similar to that reported in A. albopictus cell cultures persistently infected with Sindbis virus (R. Riedel and D. T. Brown, J. Virol. 29: 51-60, 1979) was detected in culture fluids by 3 months after infection. Bunyamwera virus replicated more rapidly in BHK cells than in mosquito cells but reached lower titers. Autointerference occurred at multiplicities of infection of 10. Virus-specific RNA and protein syntheses were at least 20% of the levels in uninfected control cells. Host cell protein synthesis was completely shut down, and nucleocapsid protein accumulated until it was 4% of the total cell protein. We discuss these results in relation to possible mechanisms involved in determining the outcome of arbovirus infection of vertebrate and mosquito cells.  相似文献   

4.
West Nile virus (WNV) is a vector-borne pathogen that was first detected in the United States in 1999. The natural transmission cycle of WNV involves mosquito vectors and avian hosts, which vary in their competency to transmit the virus. American robins are an abundant backyard species in the United States and appear to have an important role in the amplification and dissemination of WNV. In this study we examine the response of American robins to infection with various WNV doses within the range of those administered by some natural mosquito vectors. Thirty American robins were assigned a WNV dosage treatment and needle inoculated with 100.95 PFU, 101.26 PFU, 102.15 PFU, or 103.15 PFU. Serum samples were tested for the presence of infectious WNV and/or antibodies, while oral swabs were tested for the presence of WNV RNA. Five of the 30 (17%) robins had neutralizing antibodies to WNV prior to the experiment and none developed viremia or shed WNV RNA. The proportion of WNV-seronegative birds that became viremic after WNV inoculation increased in a dose dependent manner. At the lowest dose, only 40% (2/5) of the inoculated birds developed productive infections while at the highest dose, 100% (7/7) of the birds became viremic. Oral shedding of WNV RNA followed a similar trend where robins inoculated with the lower two doses were less likely to shed viral RNA (25%) than robins inoculated with one of the higher doses (92%). Viremia titers and morbidity did not increase in a dose dependent manner; only two birds succumbed to infection and, interestingly, both were inoculated with the lowest dose of WNV. It is clear that the disease ecology of WNV is a complex interplay of hosts, vectors, and viral dose delivered.  相似文献   

5.
GDVII virus growth in BHK-21 cells, a permissive host for the virus, resembled productive infections with other picornaviruses. Virus yields ranged from 100 to 600 plaque-forming units (PFU)/cell. Virus replication in HeLa cells, a nonpermissive host for GDVII virus, was characterized by virus yields of only 0.1 to 5 PFU/cell. Similar low yields of virus have been obtained from HeLa cells at all multiplicities of input up to 6,000 per cell. The progeny particles from HeLa cells were, like the infecting particles, restricted in the HeLa cell host. Despite the great difference in final yields of virus from BHK-21 and HeLa cells, the times when maximal yields were reached were similar. GDVII virus stock grown in BHK-21 cells was designated HeLa(-). A variant of GDVII virus which is capable of extensive growth in HeLa cells was obtained. This variant, designated HeLa(+) GDVII virus, was passaged serially in HeLa cells. Virus yields of 50 to 150 infective virus particles per cell were obtained from infection of HeLa cells with HeLa(+) GDVII virus. The major species of HeLa(+) virus-specific ribonucleic acid (RNA) produced was single stranded and sedimented with an S value of 35S. The rate of accumulation of HeLa(+) virus-specific RNA in HeLa cell cultures was about four times that of HeLa(-) RNA. The amount of virus-specific HeLa(+) RNA formed in HeLa cells was several-fold greater than that of HeLa(-) RNA. With HeLa(-) parent GDVII virus undergoing productive replication in BHK-21 cells or abortive replication in HeLa cells, the major species of virus-specific RNA produced was single stranded and sedimented with an approximate S value of 35S. The amount of HeLa(-) virus-specific RNA extracted from BHK-21 cells was several-fold greater than the amount obtained from HeLa cells.  相似文献   

6.
Inhibition of cellular DNA synthesis began 6 to 8 h after reovirus infection at a multiplicity of infection of 10 PFU per cell. However, as the multiplicity of infection was increased to a maximum of 103 PFU/cell, inhibition of DNA synthesis began earlier after infection (2-4 h postinfection), and the initial rate of inhibition increased. The enhanced inhibition of DNA replication at high virus multiplicities appeared to be selective since RNA synthesis was not detectably altered as late as 9 h postinfection and inhibition of protein synthesis did not begin until 7 to 9 h after infection. Early inhibition of DNA synthesis did not appear to be related to changes in thymidine pool characteristics, thymidine kinase activity, or detectable degradation of cellular DNA. Even though the particle-to-PFU ratio was increased by ultraviolet light inactivation of virus, the ability to induce early inhibition of DNA synthesis was not diminished.  相似文献   

7.
Recombinant Sendai viruses were prepared which cannot express their Cprime, C, or Cprime plus C proteins due to mutation of their respective start codons ([Cprime-minus], [C-minus] and [double mutant], respectively). The [Cprime-minus] and [C-minus] stocks were similar to that of wild-type (wt) virus in virus titer and plaque formation, whereas the double-mutant stock had a much-reduced PFU or 50% egg infective dose/particle ratio and produced very small plaques. Relative to the wt virus infection, the [Cprime-minus] and [C-minus] infections of BHK cells resulted in significantly greater accumulation of viral RNAs, consistent with the known inhibitory effects of the Cprime and C proteins. The double-mutant infection, in contrast, was delayed in its accumulation of viral RNAs; however, once accumulation started, overaccumulation quickly occurred, as in the single-mutant infections. Our results suggest that the Cprime and C proteins both provide a common positive function early in infection, so that only the double mutant undergoes delayed RNA accumulation and exhibits the highly debilitated phenotype. Later in infection, the same proteins appear to act as inhibitors of RNA accumulation. In infections of mice, [Cprime-minus] was found to be as virulent as wt virus whereas [C-minus] was highly attenuated. These results suggest that the Cprime and C proteins cannot be functionally equivalent, since C can replace Cprime for virulence in mice whereas Cprime cannot replace C.  相似文献   

8.
The GDVII strain of Theiler's murine encephalomyelitis virus (TMEV) causes an acute fatal polioencephalomyelitis in mice. Infection of susceptible mice with the DA strain of TMEV results in an acute polioencephalomyelitis followed by chronic immune-mediated demyelination with virus persistence in the central nervous system (CNS); DA virus infection is used as an animal model for multiple sclerosis. CD1d-restricted natural killer T (NKT) cells can contribute to viral clearance and regulation of autoimmune responses. To investigate the role of CD1d in TMEV infection, we first infected CD1d-deficient mice (CD1−/−) and wild-type BALB/c mice with GDVII virus. Wild-type mice were more resistant to virus than CD1−/− mice (50% lethal dose titers: wild-type mice, 10 PFU; CD1−/− mice, 1.6 PFU). Wild-type mice had fewer viral antigen-positive cells with greater inflammation in the CNS than CD1−/− mice. Second, an analysis of DA virus infection in CD1−/− mice was conducted. Although both wild-type and CD1−/− mice had similar clinical signs during the first 2 weeks after infection, CD1−/− mice had an increase in neurological deficits over those observed in wild-type mice at 3 to 5 weeks after infection. Although wild-type mice had no demyelination, 20 and 60% of CD1−/− mice developed demyelination at 3 and 5 weeks after infection, respectively. TMEV-specific lymphoproliferative responses, interleukin-4 (IL-4) production, and IL-4/gamma interferon ratios were higher in CD1−/− mice than in wild-type mice. Thus, CD1d-restricted NKT cells may play a protective role in TMEV-induced neurological disease by alteration of the cytokine profile and virus-specific immune responses.  相似文献   

9.
A total of 115 clones of Aedes albopictus cells were examined for their response to infection with Semliki Forest virus. Virus yield and cytopathology showed a bimodal distribution. More than 68% of the clones gave low yields of virus (between 8 x 10(6) and 2 x 10(8) PFU/ml) with no discernable cytopathology, and 30% gave high yields of virus (between 1 x 10(9) and 8 x 10(9) PFU/ml) and showed moderate to severe cytopathology. To determine the level at which restriction in virus growth occurs in the low-virus-producing clones, we compared the nature and extent of several virus-directed events in selected low-virus-producing clones with the same events in high-virus-producing clones. Specifically, we compared virus-specified polypeptide synthesis, positive- and negative-strand RNA synthesis, adsorption, uncoating, and transfection with virion 42S RNA. These studies showed that whereas events before negative-strand RNA synthesis and all subsequent virus-specified events were markedly reduced in the low-virus-producing lines, compared with the high-virus-producing lines. Thus, the restriction in virus growth in the low-virus-producing lines occurs at the level of synthesis of negative-strand RNA. The consequence of this restriction in an early step in the virus multiplication cycle is discussed in terms of the survival of invertebrate cells after alphavirus infection.  相似文献   

10.
Four-week-old rats (WKA/Hkm strain) were infected intranasally with the Ann Arbor/1/50 strain of influenza C virus and examined for clinical symptoms, virus replication, and serum antibody response. Although the animals showed no definite signs of illness, the virus replicated in the nose, and the hemagglutination-inhibiting (HI) and neutralizing antibodies were produced in their sera. When the inoculum sizes of 106.2 and 103.2 PFU were used, virus was recovered from nasal homogenates between days 1 and 10, and serum HI antibody became detectable by 10 days after infection. The rats infected with 101.2 PFU of the virus continued to shed virus until as late as day 20 without producing serum HI antibody. The amount of virus recovered from the nose was not affected significantly by either sex. age, or strain of the rat except that a slower virus growth was seen in the LE strain. It was also observed that the rats, previously inoculated with 103.2 PFU of the virus, showed no virus shedding when reinfected 7 weeks later but produced virus though in low titers when reinfected 50 to 55 weeks later. Virus was also recovered from rats once inoculated with 101.2 PFU of the virus when challenged 7 weeks later. Thus repeated infections characteristic of human influenza C can be produced in rats under the restricted conditions.  相似文献   

11.
Infection of non-human primates (NHPs) such as rhesus and cynomolgus macaques with monkeypox virus (MPXV) or cowpox virus (CPXV) serve as models to study poxvirus pathogenesis and to evaluate vaccines and anti-orthopox therapeutics. Intravenous inoculation of macaques with high dose of MPXV (>1–2×107 PFU) or CPXV (>102 PFU) results in 80% to 100% mortality and 66 to 100% mortality respectively. Here we report that NHPs with positive detection of poxvirus antigens in immune cells by flow cytometric staining, especially in monocytes and granulocytes succumbed to virus infection and that early positive pox staining is a strong predictor for lethality. Samples from four independent studies were analyzed. Eighteen NHPs from three different experiments were inoculated with two different MPXV strains at lethal doses. Ten NHPs displayed positive pox-staining and all 10 NHPs reached moribund endpoint. In contrast, none of the three NHPs that survived anticipated lethal virus dose showed apparent virus staining in the monocytes and granulocytes. In addition, three NHPs that were challenged with a lethal dose of MPXV and received cidofovir treatment were pox-antigen negative and all three NHPs survived. Furthermore, data from a CPXV study also demonstrated that 6/9 NHPs were pox-antigen staining positive and all 6 NHPs reached euthanasia endpoint, while the three survivors were pox-antigen staining negative. Thus, we conclude that monitoring pox-antigen staining in immune cells can be used as a biomarker to predict the prognosis of virus infection. Future studies should focus on the mechanisms and implications of the pox-infection of immune cells and the correlation between pox-antigen detection in immune cells and disease progression in human poxviral infection.  相似文献   

12.
Equine encephalosis virus (EEV) was recognized and described in the Republic of South Africa in 1967 and subsequent serological studies have shown this orbivirus to be both widespread and prevalent in southern Africa. In the present study it was shown that wild-caught Culicoides (Avaritia) imicola Kieffer (Diptera: Ceratopogonidae) can become infected with and permit the replication of the Bryanston serotype of EEV following membrane-feeding on infective blood containing 5.0 log10 plaque-forming-units (PFU)/ml. The mean prevalence of Bryanston virus infection in C. imicola after 10 days extrinsic incubation at 23.5 degrees C was 22.3% (23/103). The mean infectivity of Bryanston virus in the infected C. imicola increased from 1.3 log10 PFU/midge, in insects assayed immediately after feeding on the blood-virus mixture, to 2.6 log10 PFU/midge in insects assayed after incubation. The virus concentration in individual C. imicola infected with the Bryanston serotype of EEV ranged from 0.7 to 3.6 log10 PFU/midge. Bryanston virus titres higher than 2.5 log10 TCID50, found in individual C. imicola, suggest that this species may be able to transmit this virus to susceptible hosts. Prevalence of virus infection in C. imicola was determined by PFU and microtitration assays on both BHK and Vero cells and confirmation of the Bryanston serotype of EEV was determined by plaque inhibition. No virus replication could be demonstrated in 102 C. nivosus tested after the incubation period, suggesting that not all Culicoides species are equally susceptible to Bryanston virus infection. Other Culicoides species that survived the incubation period and that were negative for the presence of Bryanston virus were C. pycnostictus (42), C. leucostictus (7), C. magnus (2), C. bolitinos (1) and C. bedfordi (1).  相似文献   

13.
Mouse adenovirus type 1 (MAV-1) early region 1A (E1A) viral mutants were used to determine the importance of this region in pathogenesis and establishment of a persistent infection in the natural host. Lethal dose analysis with adult male Swiss outbred mice revealed a significant reduction in virulence for all of the E1A mutants. During acute infections with 105 PFU of virus, an E1A null mutant, pmE109, was found in the same organs (brain, spleen, and spinal cord) and the same cell types (endothelial cells and mononuclear cells in lymphoid tissue) as wild-type virus. Another null mutant, pmE112, was detected in the same organs but in lower numbers. However, when mice were given a lower dose, 1 PFU, pmE109 and pmE112 reached none of the target organs analyzed by 14 days postinfection (p.i.). The absence of E1A did not hinder the ability of MAV-1 to establish a persistent infection. Viral nucleic acid was detected by PCR amplification or in situ hybridization in the kidneys, brains, spleens, and prefemoral lymph nodes of mice infected with wild-type or mutant virus up to 55 weeks p.i. The brain, spleen, and lymph node are recognized sites of acute viral infection but are previously unrecognized sites for MAV-1 persistence. Evidence for the potential reactivation of persistent MAV-1 infections is also presented.  相似文献   

14.
The infectivity of intact poliovirus was not affected by exposure to the antibiotic phleomycin at concentrations as high as 200 mug/ml, whereas that of the singlestranded poliovirus ribonucleic acid (RNA) was inactivated to 99% by pretreatment of the RNA with phleomycin at a concentration of 2 mug/ml. The infectivity of double and multistranded RNA was 10 times less sensitive than that of singlestranded RNA to the action of this antibiotic. Preincubation of HeLa cells for 30 min with 10 to 50 mug of phleomycin reduced the sensitivity of the cells to infection by viral RNA and intact virus, indicating that phleomycin interferes with cellular functions necessary for virus replication. When phleomycin was added to cells at different times after infection with single- or double-stranded RNA, the highest inactivation of infective centers was observed immediately after infection. With time of incubation at 37 C, the infective centers became more resistant to the action of phleomycin.  相似文献   

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The production of foot-and-mouth disease (FMD) virus in baby hamster kidney (BHK) suspension cells grown in serum-free media for subsequent use in vaccines was attempted because of the limited availability of serum in quantities sufficient for propagation of large amounts of cells, as well as the possible presence of mycoplasma, viral contaminants, and interfering antibodies in sera. Suspension cultures (50 to 600 ml) of BHK-21 cells adapted to and continually passed in a glutamine-free autoclavable, chemically defined medium (BHK-S system) were infected with all seven types of FMD virus. Cells were infected at multiplicities of infection (MOI) ranging from 10?1 to 10?7 plaque-forming units per cell (PFU/cell). The time course of infectious virus release and the amount of complement-fixing (CF) antigen produced were then followed. Peak harvest infectivities of approximately 108.5 PFU/ml were obtained from 12 to 24 hr after inoculation, depending on input MOI, and were apparently independent of cell concentration over the range 1.5 to 4.0 million cells/ml; the CF endpoint dilutions increased from 1:12 at the lower cell concentrations to 1:48 at the highest cell concentration. Monovalent and trivalent vaccines have been produced using viruses from the BHK-S system, inactivated with acetylethyleneimine and emulsified in oil, and the results of tests in steers and guinea pigs are presented.  相似文献   

17.
A stable line of baby hamster kidney cells for use in the production of, and subsequent purification of, foot-and-mouth disease virus (FMDV) was grown in large quantities on the cylindrical surfaces of 2-liter Baxter bottles. The bottles, in round wire cages, were rotated on a three-tiered roller mill. The cells retained their rapid growth characteristics and susceptibility to FMDV in a tris(hydroxymethyl)aminomethane buffer-containing medium which was especially formulated for large-scale work. This medium, without being changed, sustained cell growth for 6 to 7 days to yield confluent layers containing 500 to 750 million cells per bottle. In small-scale virus-growth experiments, harvested fluids contained about 103.8 to 108.8 plaque-forming units (PFU) per ml. This corresponded to a yield of 30 to 50 PFU per cell. In production runs with 190 cultures, the infectious fluids usually contained 107.9 to 109.2 PFU per ml, and the mass of essentially pure virus obtained therefrom ranged from 7 to 17 mg concomitant with cumulative infectivity recoveries of about 20%.  相似文献   

18.
Cricket paralysis virus, an insect picorna-like virus, grown and assayed on Drosophila malanogaster cells, gave titers in excess of 109 PFU/ml. The virus was plaque purified using an agar overlay and the intracellular polypeptides induced by “crude” and plaque-purified virions were demonstrated to be very similar.  相似文献   

19.
Untreated rabies virus (RABV) infection leads to death. Vaccine and postexposure treatment have been effective in preventing RABV infection. However, due to cost, rabies vaccination and treatment have not been widely used in developing countries. There are 55,000 human death caused by rabies annually. An efficacious and cost-effective rabies vaccine is needed. Parainfluenza virus 5 (PIV5) is thought to contribute to kennel cough, and kennel cough vaccines containing live PIV5 have been used in dogs for many years. In this work, a PIV5-vectored rabies vaccine was tested in mice. A recombinant PIV5 encoding RABV glycoprotein (G) (rPIV5-RV-G) was administered to mice via intranasal (i.n.), intramuscular (i.m.), and oral inoculation. The vaccinated mice were challenged with a 50% lethal challenge dose (LD50) of RABV challenge virus standard 24 (CVS-24) intracerebrally. A single dose of 106 PFU of rPIV5-RV-G was sufficient for 100% protection when administered via the i.n. route. The mice vaccinated with a single dose of 108 PFU of rPIV5-RV-G via the i.m. route showed very robust protection (90% to 100%). Intriguingly, the mice vaccinated orally with a single dose of 108 PFU of rPIV5-RV-G showed a 50% survival rate, which is comparable to the 60% survival rate among mice inoculated with an attenuated rabies vaccine strain, recombinant LBNSE. This is first report of an orally effective rabies vaccine candidate in animals based on PIV5 as a vector. These results indicate that rPIV5-RV-G is an excellent candidate for a new generation of recombinant rabies vaccine for humans and animals and PIV5 is a potential vector for oral vaccines.  相似文献   

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