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Assessment of the lysis efficiency of selected guanidinium thiocyanate/hydrochloride lysis buffers commonly used in PCR diagnostics
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Introduction
Bovine coronavirus (BCoV) is a causative agent of enteric and respiratory diseases in cattle. Despite its importance for animal health, no data is available on its prevalence in Poland. The aim of the study was to determine the virus’ seroprevalence, identify risk factors of BCoV exposure in selected cattle farms and investigate the genetic variability of circulating strains.
Material and Methods
Serum and nasal swab samples were collected from 296 individuals from 51 cattle herds. Serum samples were tested with ELISA for the presence of BCoV-, bovine herpesvirus-1 (BoHV-1)- and bovine viral diarrhoea virus (BVDV)-specific antibodies. The presence of those viruses in nasal swabs was tested by real-time PCR assays. Phylogenetic analysis was performed using fragments of the BCoV S gene.
Results
Antibodies specific to BCoV were found in 215 (72.6%) animals. Seropositivity for BCoV was more frequent (P>0.05) in calves under 6 months of age, animals with respiratory signs coinfected with BoHV-1 and BVDV and increased with herd size. In the final model, age and herd size were established as risk factors for BCoV-seropositivity. Genetic material of BCoV was found in 31 (10.5%) animals. The probability of BCoV detection was the highest in medium-sized herds. Polish BCoVs showed high genetic homology (98.3–100%) and close relatedness to European strains.
Conclusion
Infections with BCoV were more common than infections with BoHV-1 and BVDV. Bovine coronavirus exposure and shedding show age- and herd density-dependence.
The emergence and sustained transmission of novel pathogens are exerting an increasing demand on the diagnostics sector worldwide, as seen with the ongoing SARS‐CoV‐2 pandemic and the more recent public health concern of monkeypox virus (MPXV) since May 2022. Appropriate and reliable viral inactivation measures are needed to ensure the safety of personnel handling these infectious samples. In the present study, 7 commercialized diagnosis buffers, heat [56°C and 60°C], and sodium dodecyl sulfate detergent [SDS; 2.0%, 1.0%, and 0.5% final concentrations] were tested against infectious SARS‐CoV‐2 and MPXV culture isolates on Vero cell culture. Cytopathic effects were observed up to 7 days post‐inoculation and viral load evolution was measured by semi‐quantitative PCR. WHO recommends an infectious titer reduction of at least 4 log10. As such, the data show efficacious SARS‐CoV‐2 inactivation by all investigated methods, with >6.0 log10 reduction. MPXV inactivation was also validated with all investigated methods with 6.9 log10 reduction, although some commercial buffers required a longer incubation period to yield complete inactivation. These results are valuable for facilities, notably those without BSL‐3 capabilities, that need to implement rapid and reliable protocols common against both SARS‐CoV‐2 and MPXV. This article is protected by copyright. All rights reserved.
The objective of this study was to determine the inactivation efficiency of common sample preparation reagents against highly pathogenic avian influenza A (HPAI) H5N1 virus. HPAI H5N1 virus has caused infections in humans with a mortality rate of over 50%. Due to the high mortality and the risk of aerosol transmission of that virus to humans and birds, infectious HPAI H5N1 viruses are contained in a biosafety level 3 laboratory. However, many procedures for further molecular analyses would be easier in lower biosafety conditions. To ensure the laboratory safety the successful inactivation procedures should be demonstrated before the samples are transferred to a lower containment facility.
We tested the inactivation capacity of commonly used cell lysis buffer radio-immuno precipitation assay (RIPA) buffer for protein samples, cell fixatives methanol (MeOH) and paraformaldehyde (PFA) and guanidine isothiocyanate-containing lysis buffer for RNA isolation (RLT, Qiagen) in H5N1-infected cells. Based on our results RLT buffer, 90% MeOH (20 min, -20°C) and 4% PFA (30 min, RT) all completely inactivated the HPAI H5N1 virus. However, RIPA buffer alone was not sufficient to inactivate the HPAI H5N1 virus in infected cell samples but, instead, combining RIPA lysis buffer and boiling for 10 min the samples in Laemmli buffer led to complete inactivation of the virus.
The handling of highly pathogenic viruses, whether for diagnostic or research purposes, often requires an inactivation step. This article reviews available inactivation techniques published in peer-reviewed journals and their benefits and limitations in relation to the intended application. The bulk of highly pathogenic viruses are represented by enveloped RNA viruses belonging to the Togaviridae, Flaviviridae, Filoviridae, Arenaviridae, Hantaviridae, Peribunyaviridae, Phenuiviridae, Nairoviridae and Orthomyxoviridae families. Here, we summarize inactivation methods for these virus families that allow for subsequent molecular and serological analysis or vaccine development. The techniques identified here include: treatment with guanidium-based chaotropic salts, heat inactivation, photoactive compounds such as psoralens or 1.5-iodonaphtyl azide, detergents, fixing with aldehydes, UV-radiation, gamma irradiation, aromatic disulfides, beta-propiolacton and hydrogen peroxide. The combination of simple techniques such as heat or UV-radiation and detergents such as Tween-20, Triton X-100 or Sodium dodecyl sulfate are often sufficient for virus inactivation, but the efficiency may be affected by influencing factors including quantity of infectious particles, matrix constitution, pH, salt- and protein content. Residual infectivity of the inactivated virus could have disastrous consequences for both laboratory/healthcare personnel and patients. Therefore, the development of inactivation protocols requires careful considerations which we review here.
Clinical samples collected in coronavirus disease 19 (COVID-19), patients are commonly manipulated in biosafety level 2 laboratories for molecular diagnostic purposes. Here, we tested French norm NF-EN-14476+A2 derived from European standard EN-14885 to assess the risk of manipulating infectious viruses prior to RNA extraction. SARS-CoV-2 cell-culture supernatant and nasopharyngeal samples (virus-spiked samples and clinical samples collected in COVID-19 patients) were used to measure the reduction of infectivity after 10 minute contact with lysis buffer containing various detergents and chaotropic agents. A total of thirteen protocols were evaluated. Two commercially available formulations showed the ability to reduce infectivity by at least 6 log 10, whereas others proved less effective.
Coronaviruses (CoVs) belong to the family of Coronaviridae, the order Nidovirales, and the genus Coronavirus. They are the largest group of viruses causing respiratory and gastrointestinal infections. Morphologically, CoVs are enveloped viruses containing a non-segmented positive-sense, single-stranded ribonucleic acid (RNA) viruses. CoVs are categorized into four important genera that include Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. A novel member of human CoV that has recently emerged in Wuhan, China, is now formally named as SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). This is a unique strain of RNA viruses that have not been previously observed in humans. The virus has wide host adaptability and is capable of causing severe diseases in humans, masked palm civets, mice, dogs, cats, camels, pigs, chickens, and bats. The SARS-CoV-2 typically causes respiratory and gastrointestinal sickness in both humans and animals. It can be transmitted through aerosols and direct/indirect contact, as well as during medical cases and laboratory sample handling. Specific structural proteins, which might be found on the surface of the virus, play an important role in the pathogenesis and development of the complications. The disease is characterized by distinct medical signs and symptoms that include high fever, chills, cough, and shortness of breath or difficulty in breathing. The infected people may also present with other symptoms such as diarrhea, myalgia, fatigue, expectoration, and hemoptysis. It is important from the public health and economic point of view as it affects the growth of the country, which is majorly attributed to the restriction in the movement of the people and the cost associated with the control and prevention of the disease. Since there is no specific therapeutic intervention nor a vaccine available against the virus, supportive management and treatment with non-specific therapeutic agents (repurposed drugs) may provide relief to the patients. Some preventive strategies of the disease include blocking the routes of transmission of the infections, disinfection of instruments used during medical case handling, using personal protective equipment, proper and early diagnosis of the disease, avoiding contact with the sick patients, and quarantine of the infected/exposed people.
Laboratories working with foot-and-mouth disease virus (FMDV) must maintain a high level of biocontainment. However, if infectious virus is reliably inactivated during sample processing, molecular and serological testing can be performed at a lower level of containment. In this study, three commercial lysis buffers (AL, AVL, and MagMAX CORE) were tested in two laboratories for their ability to inactivate FMDV A/IRN/8/2015 in different sample matrices (cell culture supernatant, epithelial tissue suspension and milk). Residual infectivity after the addition of lysis buffer was evaluated by inoculating susceptible cell cultures. No cytopathic effect was observed for all three lysis buffers, indicating that the buffers are capable of reducing viral infectivity (estimated range 3.1 to >5.1 Log10). These results highlight the capacity of lysis buffers to decrease FMDV infectivity; however, additional validation experiments should be conducted, particularly if different sample matrices and/or lysis buffers are used.
Objectives:
To assess the prevalence of canine parainfluenza virus, canine adenovirus type 2, canine distemper virus, canine respiratory coronavirus and influenza virus A infections in: (1) privately-owned or, (2) kennelled dogs showing signs consistent with canine infectious respiratory disease and, (3) clinically healthy dogs in Vienna, Austria.
Materials and methods:
Prospectively, nasal and tonsillar swabs from 214 dogs affected with infectious respiratory disease, and 50 healthy control dogs were tested for nucleic acids specific to the various viral infections. Concurrent bronchoalveolar lavage fluid from 31 dogs with chronic respiratory disease was investigated for the same viral pathogens. Additionally, anti-canine respiratory coronavirus antibody concentrations were measured in paired blood samples from 30 acutely diseased dogs.
Results:
Canine respiratory coronavirus (7.5%) and canine parainfluenza virus (6.5%) were the most commonly detected viruses in samples from the upper airways of dogs with respiratory infections. Serological results showed a significant seroconversion in response to coronavirus in 50% of the examined cases. None of the samples was positive for influenza virus A-specific nucleic acid. Canine coronavirus-specific nucleic acid was detected in 4.0% of healthy dogs.
Clinical significance:
Canine coronavirus should be considered as a clinically relevant cause of infectious respiratory disease in crowded dog populations. For sample collection, the nasal mucosa can be recommended as the favoured site. Analysis of paired serum samples aids verification of canine coronavirus infection in respiratory disease.
Introduction
Albendazole is used to treat endoparasitic diseases in animals and humans. After oral administration, it is quickly oxidised into its pharmacologically active metabolite albendazole sulfoxide and then to sulfone. However, it is not clear which compound is responsible for toxic effects towards mammalian cells.
Material and Methods
The model systems comprised cultures of isolated rat hepatocytes, two hepatoma cell lines (FaO, HepG2), and non-hepatic Balb/c 3T3 line. Cells were exposed for 24, 48, and 72 h to eight concentrations of albendazole ranging from 0.05 to 100 µg/mL. At all three time points cytotoxic effects were assessed by MTT assay and metabolites in the culture media were determined by LC-MS/MS analysis.
Results
The effective concentrations EC50-72h showed that Balb/c 3T3 cells were the most sensitive to albendazole (0.2 ±0.1 µg/mL) followed by FaO (1.0 ±0.4 µg/mL), and HepG2 (6.4 ±0.1 µg/mL). In the case of isolated hepatocytes this value could not be attained up to the highest concentration used. Chemical analysis revealed that the concentrations of albendazole in hepatocytes and HepG2 and FaO culture media gradually decreased with incubation time, while the concentrations of its metabolites increased. The metabolism in isolated hepatocytes was dozens of times greater than in HepG2 and FaO cells. Two metabolites (albendazole sulfoxide, albendazole sulfone) were detected in isolated hepatocytes and HepG2 culture medium, one (albendazole sulfoxide) in FaO culture medium and none in Balb/c 3T3.
Conclusion
The obtained data indicate that metabolism of albendazole leads to its detoxification. The lower cytotoxic potential of metabolites was confirmed in the independent experiments in this study.
Rapid bedside inactivation of Ebola virus would be a solution for the safety of medical and technical staff, risk containment, sample transport and high-throughput or rapid diagnostic testing during an outbreak. We show that the commercially available MagNA Pure lysis/binding buffer used for nucleic acid extraction inactivates Ebola virus. A rapid bedside inactivation method for nucleic acid tests is obtained by simply adding MagNA Pure lysis/binding buffer directly into vacuum blood collection EDTA-tubes using a thin needle and syringe prior to sampling. The ready-to-use inactivation vacuum tubes are stable for more than 4 months and Ebola virus RNA is preserved in the MagNA Pure lysis/binding buffer for at least 5 weeks independent of the storage temperature. We also show that Ebola virus RNA can be manually extracted from MagNA Pure lysis/binding buffer-inactivated samples using the QIAamp Viral RNA mini kit. We present an easy and convenient method for bedside inactivation using available blood collection vacuum tubes and reagents. We propose to use this simple method for fast, safe and easy bedside inactivation of Ebola virus for safe transport and routine nucleic acid detection.
Reliable inactivation of specimens before removal from high-level biocontainment is crucial for safe operation. To evaluate efficacy of methods of chemical inactivation, we compared in vitro and in vivo approaches using Ebola virus as a surrogate pathogen. Consequently, we have established parameters and protocols leading to reliable and effective inactivation.
Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a recently emerged virus that has caused a number of human infections and deaths, primarily in the Middle East. The transmission of MERS-CoV to humans has been proposed to be as a result of contact with camels, but evidence of human-to-human transmission also exists. In order to work with MERS-CoV in a laboratory setting, the US Centers for Disease Control and Prevention (CDC) has determined that MERS-CoV should be handled at a biosafety level (BSL) 3 (BSL-3) biocontainment level. Many processes and procedures used to characterize MERS-CoV and to evaluate samples from MERS-CoV infected animals are more easily and efficiently completed at BSL-2 or lower containment. In order to complete experimental work at BSL-2, demonstration or proof of inactivation is required before removal of specimens from biocontainment laboratories. In the studies presented here, we evaluated typical means of inactivating viruses prior to handling specimens at a lower biocontainment level. We found that Trizol, AVL buffer and gamma irradiation were effective at inactivating MERS-CoV, that formaldehyde-based solutions required at least 30min of contact time in a cell culture system while a mixture of methanol and acetone required 60min to inactivate MERS-CoV. Together, these data provide a foundation for safely inactivating MERS-CoV, and potentially other coronaviruses, prior to removal from biocontainment facilities.
Copyright © 2015 Elsevier B.V. All rights reserved.
Rapid inactivation of Ebola Virus (EBOV) is crucial for high-throughput testing of clinical samples in low-resource, outbreak scenarios. The EBOV inactivation efficacy of Buffer AVL (Qiagen) was tested against marmoset serum (EBOV concentration of 1 × 10(8) TCID50⋅mL(-1)) and murine blood (EBOV concentration of 1 × 10(7) TCID50⋅mL(-1)) at 4:1 v/v buffer:sample ratios. Post-treatment cell culture and ELISA analysis indicated that treatment with Buffer AVL did not inactivate EBOV in 67% of samples, indicating that Buffer AVL, which is designed for RNA extraction and not virus inactivation, cannot be guaranteed to inactivate EBOV in diagnostic samples. Murine blood samples treated with ethanol (4:1 [v/v] ethanol:sample) or heat (60(o)C: 15 min) also showed no viral inactivation in 67% or 100% of samples respectively. However, combined Buffer AVL + ethanol or Buffer AVL + heat treatments showed total viral inactivation in 100% samples tested. The Buffer AVL + ethanol and Buffer AVL + heat treatments were also shown not to affect the extraction of PCR quality RNA from EBOV spiked murine blood samples.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
An epidemiological survey for Canine parvovirus 2 (CPV-2) and Canine coronavirus (CCoV) was conducted in Albania. A total of 57 fecal samples were collected from diarrheic dogs in the District of Tirana during 2011-2013. The molecular assays detected 53 and 31 CPV- and CCoV-positive specimens, respectively, with mixed CPV-CCoV infections diagnosed in 28 dogs. The most frequently detected CPV type was 2a, whereas IIa was the predominant CCoV subtype. A better comprehension of the CPV-CCoV epidemiology in eastern European countries will help to assess the most appropriate vaccination strategies to prevent disease due to infections with these widespread agents of acute gastroenteritis in the dog.
During the past years, human coronaviruses (HCoVs) have been increasingly identified as pathogens associated with more-severe
respiratory tract infection (RTI). Diagnostic tests for HCoVs are not frequently used in the routine setting. It is likely
that, as a result, the precise role that HCoVs play in RTIs is greatly underestimated. We describe a rapid, sensitive, and
highly specific quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR) for the detection of HCoV
that can easily be implemented in the routine diagnostic setting. HCoV was detected in 28 (11%) of the 261 clinical specimens
obtained from patients presenting with symptoms of RTI ranging from common cold to severe pneumonia. Only 1 (0.4%) of the
243 control specimens obtained from patients without symptoms of RTI showed the presence of HCoV. We conclude that HCoVs can
be frequently detected in patients presenting with RTI. Real-time RT-PCR provides a tool for large-scale epidemiological studies
to further clarify the role that coronavirus infection plays in RTI in humans.
Hepatitis C virus (HCV) infection is an increasing health problem worldwide. Quantitative assays for HCV viral load are valuable
in predicting response to therapy and for following treatment efficacy. Unfortunately, most quantitative tests for HCV RNA
are limited by poor sensitivity. We have developed a convenient, highly sensitive real-time reverse transcription-PCR assay
for HCV RNA. The assay amplifies a portion of the 5′ untranslated region of HCV, which is then quantitated using the TaqMan
7700 detection system. Extraction of viral RNA for our assay is fully automated with the MagNA Pure LC extraction system (Roche).
Our assay has a 100% detection rate for samples containing 50 IU of HCV RNA/ml and is linear up to viral loads of at least
109 IU/ml. The assay detects genotypes 1a, 2a, and 3a with equal efficiency. Quantitative results by our assay correlate well
with HCV viral load as determined by the Bayer VERSANT HCV RNA 3.0 bDNA assay. In clinical use, our assay is highly reproducible,
with high and low control specimens showing a coefficient of variation for the logarithmic result of 2.8 and 7.0%, respectively.
The combination of reproducibility, extreme sensitivity, and ease of performance makes this assay an attractive option for
routine HCV viral load testing.
An unidentified agent was cultured in primary monkey cells at the Los Angeles County Public Health Department from each of
five stool specimens submitted from an outbreak of gastroenteritis. Electron microscopy and an adenovirus-specific monoclonal
antibody confirmed this agent to be an adenovirus. Since viral titers were too low, complete serotyping was not possible.
Using the DNase-sequence-independent viral nucleic acid amplification method, we identified several nucleotide sequences with
a high homology to human adenovirus 41 (HAdV-41) and simian adenovirus 1 (SAdV-1). However, using anti-SAdV-1 sera, it was
determined that this virus was serologically different than SAdV-1. Genomic sequencing and phylogenetic analysis confirmed
that this new adenovirus was so divergent from the known human adenoviruses that it was not only a new type but also represented
a new species (human adenovirus G). In a retrospective clinical study, this new virus was detected by PCR in one additional
patient from a separate gastroenteritis outbreak. This study suggests that HAdV-52 may be one of many agents causing gastroenteritis
of unknown etiology.
The efforts of the Global Poliovirus Eradication Initiative (GPEI) have brought about the near elimination of poliovirus worldwide. The World Health Organization has issued guidelines for the safe handling and containment of infectious materials (IM) and potentially infectious materials (PIM) following poliovirus eradication. Inactivation of poliovirus in IM and PIM is needed to prevent inadvertent re-introduction of polioviruses post-eradication. In this study, we investigated the use of guanidine thiocyanate-based nucleic acid extraction buffers from commercially available nucleic acid extraction kits to inactivate poliovirus in cell culture isolates and stool suspensions, two common types of poliovirus IM and PIM, respectively. Incubation with selected nucleic acid extraction buffers or extraction buffers supplemented with ethanol reduced the infectivity of high-titer wild poliovirus type 1 (WPV1), wild poliovirus type 3 (WPV3), Sabin 1 (SL1), and Sabin 3 (SL3) cell culture isolates below the limit of detection in CCID50 assays. Stool suspensions containing WPV1, WPV3, SL1, SL2, or SL3 were also inactivated by the extraction buffers tested. Blind passage of WPV1-spiked stool suspensions confirmed complete inactivation of WPV1 after incubation with extraction buffers. Moreover, treatment with a buffer consisting of 4 M guanidine thiocyanate with 30% ethanol inactivated a high-titer WPV1 culture isolate and a WPV1-spiked stool suspension. Taken together, these results show that guanidine thiocyanate-based nucleic acid extraction buffers are an effective means of inactivating poliovirus IM and PIM, and thus will be instrumental in ensuring containment compliance and preventing potential re-emergence of contained polioviruses.
Background:
Corona Virus Disease-2019 (COVID-19) has spread widely throughout the world since the end of 2019. Nucleic acid testing (NAT) has played an important role in patient diagnosis and management of COVID-19. In some circumstances, thermal inactivation at 56 °C has been recommended to inactivate Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) before NAT. However, this procedure could theoretically disrupt nucleic acid integrity of this single-stranded RNA virus and cause false negatives in real-time polymerase chain reaction (RT-PCR) tests.
Methods:
We investigated whether thermal inactivation could affect the results of viral NAT. We examined the effects of thermal inactivation on the quantitative RT-PCR results of SARS-CoV-2 particularly with regard to the rates of false-negative results for specimens carrying low viral loads. We additionally investigated the effects of different specimen types, sample preservation times and a chemical inactivation approach on NAT.
Results:
Our work showed increased Ct values in specimens from diagnosed COVID-19 patients in RT-PCR tests after thermal incubation. Moreover, about half of the weak-positive samples (7 of 15 samples, 46.7%) were RT-PCR negative after heat inactivation in at least one parallel testing. The use of guanidinium-based lysis for preservation of these specimens had a smaller impact on RT-PCR results with fewer false negatives (2 of 15 samples, 13.3%) and significantly less increase in Ct values than heat inactivation.
Conclusion:
Thermal inactivation adversely affected the efficiency of RT-PCR for SARS-CoV-2 detection. Given the limited applicability associated with chemical inactivators, other approaches to ensure the overall protection of laboratory personnel need consideration.
Working with virological samples requires validated inactivation protocols for safe handling and disposal. Although many techniques exist to inactivate samples containing viruses, not all procedures have been properly validated or are compatible with subsequent assays. To aid in the development of inactivation protocols for Alphaviruses, and specifically Venezuelan equine encephalitis virus (VEEV), a variety of methods were evaluated for their ability to completely inactivate a high titer sample of the vaccine strain VEEV TC-83. The methods evaluated include reagents used in RNA extraction, fixation, treatment with a detergent, and heat inactivation. Most methods were successful at inactivating the sample; however, treatment with only Buffer AVL, SDS, and heat inactivation at 58 °C for one hour were not capable of complete inactivation of the virus in the sample. These results provide a substantial framework for identifying techniques that are safe for complete inactivation of Alphaviruses and to advise protocol implementation.
The 2014 Ebola outbreak in West Africa required the rapid testing of clinical material for the presence of potentially high titre Ebola virus (EBOV). Safe, fast and effective methods for the inactivation of such clinical samples are required so that rapid diagnostic tests including downstream analysis by RT-qPCR or nucleotide sequencing can be carried out. One of the most commonly used guanidinium - based denaturing agents, AVL (Qiagen) has been shown to fully inactivate EBOV once ethanol is added, however this is not compatible with the use of automated nucleic acid extraction systems. Additional inactivation agents need to be identified that can be used in automated systems. A candidate inactivation agent is Triton X-100, a non-denaturing detergent that is frequently used in clinical nucleic acid extraction procedures and has previously been used for inactivation of EBOV. In this study the effect of 0.1% and 1.0% Triton X-100 (final concentration 0.08% and 0.8% respectively) alone and in combination with AVL on the viability of EBOV (10(6) TCID50/ml) spiked into commercially available pooled negative human serum was tested. The presence of viable EBOV in the treated samples was assessed by carrying out three serial passages of the samples in Vero E6 cells (37°C, 5% CO2, 1 week for each passage). At the end of each passage the cells were observed for evidence of cytopathic effect and samples were taken for rRT-PCR analysis for the presence of EBOV RNA. Before cell culture cytotoxic components of AVL and Triton X-100 were removed from the samples using size exclusion spin column technology or a hydrophobic adsorbent resin. The results of this study showed that EBOV spiked into human serum was not fully inactivated when treated with either 0.1% (v/v) Triton X-100 for 10 mins or 1.0% (v/v) Triton X-100 for 20 mins (final concentrations 0.08% and 0.8% Triton X-100 respectively). AVL alone also did not consistently provide complete inactivation. Samples treated with both AVL and 0.1% Triton X-100 for 10 or 20 mins were shown to be completely inactivated. This treatment is compatible with downstream analysis by RT-qPCR and next generation sequencing.
There is a common assumption that viral lysis buffers are sufficient to render viruses noninfectious. This assumption has a significant impact on the way biological samples are processed, labeled, and handled for shipment. Several lysis buffers, including TRIzol, AVL, RLT, MagMAX, and easyMAG, were examined for their capacity to inactivate representative viruses from multiple genera, including alphavirus, bunyavirus, flavivirus, adenovirus, enterovirus, influenza B, and simplexvirus. Viruses were noninfectious following treatment with TRIzol, MagMAX, and easyMAG buffers, while some viruses were still viable in cell cultures following treatment with AVL and RLT buffers. These results indicate the need to further evaluate the expectation that lysis buffers render live viruses inactive, allowing handling and processing of these samples under low-level containment conditions.
Hepatitis A virus infections have been reported recently among hemophilic patients in Italy and Germany, leading to speculation that infectious hepatitis A virus (HAV) might have been present in some factor VIII concentrates. In both cases, the implicated factor concentrates had been treated by a solvent/detergent method, which inactivates enveloped viruses but which would not be expected to inactivate HAV, a nonenveloped picornavirus. To determine whether HAV would be inactivated during pasteurization of factor VIII concentrate, an alternative method employed for virus inactivation, we determined the extent to which the infectivity of cell culture-adapted HAV, suspended either in cell culture medium or in a proprietary stabilizing buffer, was reduced by heat treatment at 60°C for 10 hr. The titer of infectious HAV declined rapidly at 60°C, but the stabilizer considerably delayed HAV inactivation. In cell culture medium, HAV was inactivated by >3.6 log10 within 30 min, but 3.6 log10 inactivation of HAV was reached only after 6 hr in the presence of the stabilizer. Residual infectious HAV was present after even 10 hr of heat treatment in the stabilizer, indicating that <5.2 log10 infectious HAV particles are inactivated under these conditions. In the presence of the stabilizer, HAV was significantly more stable than poliovirus type 1, which has been used to validate virus inactivation by pasteurization. We conclude that pasteurized factor VIII concentrate should pose little if any risk for transmission of HAV if pooled plasma used for its manufacture contained low levels of the virus.
Background and aims: Canine adenovirus type 2 (CAV-2) infection is typically diagnosed histopathologically since intranuclear inclusion bodies (IN/IBs) are demonstrable in the infected lung. However, it is sometimes difficult to identify IN/IBs, particularly in autolyzed tissues or samples from both early and late stages of infection, and other methods were presently developed. Methods: Stray dog samples were evaluated by histopathology, polymerase chain reaction (PCR), and immunohistochemistry (IHC) to investigate the status of the CAV-2 infection on the stray dogs in Korea. Histologic tests were performed, and dogs with pneumonic lungs were further evaluated by IHC and PCR. Results: Pathognomonic IN/IBs were identified in 3 of 213 lungs; CAV-2 PCR was positive for 27 of 213 pneumonic lungs. A total of 7 of 27 CAV-2 PCR-positive lungs were IHC-positive. No PCR-negative lung was IHC-positive. Positive results were primarily detected in the IN/IBs of the bronchial epithelial cells, macrophages, and very rarely in the cytoplasm of bronchial epithelial cells. Conclusions: IHC was a more reliable diagnostic method than conventional pathologic methods in the present study, and suggests that IHC should be routinely used in the diagnosis of CAV-2 infection. Further, PCR alone may not be adequate for CAV-2 diagnosis.
Es wird ein Verfahren zur kollektiven Behandlung von Reihenversuchen angegeben, das gestattet, auch bei kleinerem Umfang des Kollektivs (Reihenversuch mit vier bis fnf Gruppen zu je sechs Tieren) und bei starker Streuung zu einem zahlenmigen Ausdruck des Versuchsergebnisses zu gelangen.
To clarify the prevalence of canine coronavirus (CCoV) infection in Japan, faecal samples from 109 dogs with diarrhoea were examined for CCoV RNA together with canine parvovirus type 2 (CPV-2) DNA. The detection rates of CCoV and CPV-2 for dogs aged less than 1 year were 66.3% and 43.8%, while those for dogs aged 1 year or older were 6.9% and 10.3%, respectively, which were significantly different (p<0.0001 and p=0.0003, respectively), indicating not CPV-2 but CCoV is an important diarrhoea-causing organism in juvenile dogs. Among the CCoV-positive dogs, 65.5% and 72.7% showed to be positive for CCoV types I and II, respectively, and simultaneous detection rate of both types was high at 40.0%. Furthermore, transmissible gastroenteritis virus (TGEV)-like CCoV RNA was detected from 8 dogs. These findings indicate that CCoV type I and TGEV-like CCoV are already circulating in Japan, though no reports have been presented to date.
The stabilities of hepatitis A virus (HAV) and of poliovirus type 2 were compared under strictly controlled, identical conditions of pH value, temperature, and salt concentration. Although the resistance of the viruses proved to be the same from pH 3 to 11, the temperature at which 50% of poliovirus particles became disintegrated during heating at pH 7.0 for 10 min (T50,10 = 43 degrees) differed significantly from that characteristic for HAV (T50,10 = 61 degrees). In the presence of 1 M MgCl2, the T50,10 for poliovirus and for HAV shifted to 61 degrees and 81 degrees, respectively. Destabilization of the physical integrity of HAV by heating resulted in the release of viral RNA and, simultaneously, in the generation of several 'empty' capsid structures or dissociation products thereof. Empty capsids and further dissociation products were still serologically reactive with anti-HAV IgG contained in human convalescent sera. With further heating (greater than T50,10 = 67 degrees or greater than T50,1 = 87 degrees), however, they irreversibly lost this reactivity.
A tetrazolium salt has been used to develop a quantitative colorimetric assay for mammalian cell survival and proliferation. The assay detects living, but not dead cells and the signal generated is dependent on the degree of activation of the cells. This method can therefore be used to measure cytotoxicity, proliferation or activation. The results can be read on a multiwell scanning spectrophotometer (ELISA reader) and show a high degree of precision. No washing steps are used in the assay. The main advantages of the colorimetric assay are its rapidity and precision, and the lack of any radioisotope. We have used the assay to measure proliferative lymphokines, mitogen stimulations and complement-mediated lysis.
Many assume that common methods to extract viral nucleic acids are able to render a sample non-infectious. It may be that inactivation of infectious virus is incomplete during viral nucleic acid extraction methods. Accordingly, two common viral nucleic acid extraction techniques were evaluated for the ability to inactivate high viral titer specimens. In particular, the potential for TRIzol LS Reagent (Invitrogen Corp., Carlsbad, CA) and AVL Buffer (Qiagen, Valencia, CA) were examined to render suspensions of alphaviruses, flaviviruses, filoviruses and a bunyavirus non-infectious to tissue culture assay. The dilution series for both extraction reagents consistently caused cell death through a 100-fold dilution. Except for the DEN subtype 4 positive control, all viruses had titers of at least 10(6)pfu/ml. No plaques were detected in any extraction reagent plus virus combination in this study, therefore, the extraction reagents appeared to inactivate completely each of the high-titer viruses used in this study. These results support the reliance upon either TRIzol LS Reagent or AVL Buffer to render clinical or environmental samples non-infectious, which has implications for the handling and processing of samples under austere field conditions and low level containment.
A method was developed for detection of hepatitis A virus (HAV) in soft fruits (raspberries and strawberries). After washing the sample in 1 M sodium bicarbonate with added soya protein, fruits were removed by slow speed centrifugation, then particulate material and residual pectin were removed from the supernatant by flocculation and pectinase treatment during another slow speed centrifugation. Virus particles were then sedimented by ultracentrifugation. RNA was extracted from the virus particles, and nested RTPCR was performed on the nucleic acid extract. Nested RTPCR comprised an RTPCR, followed by PCR to amplify sequences within the amplicon. Internal amplification controls (IACs) were constructed for both the RTPCR and the PCR. The sensitivity of the nested RTPCR was approximately 10 RTPCRU. The overall method was shown to be able to detect 10(4) RTPCRU HAV in 90 g fresh strawberries, and 10(3) RTPCRU HAV in 60 g fresh raspberries. It is estimated that the lowest possible limit of detection of the method should be between 40 and 400 RTPCRU HAV per fruit sample. The method can be performed within one day, in suitably equipped microbiological laboratories, and is suitable for routine screening of food samples, and for analysis of suspected samples, e.g. during outbreak investigations.
Diagnoses of ongoing viral infections commonly rely on PCR methodology. Sample material that may contain hazardous virus should be efficiently inactivated in biological containment or bed-side before diagnostic PCR analysis. Surprisingly little documentation is available for inactivation of human viral pathogens by inactivation reagents that allow for subsequent PCR diagnostics. It is now shown that pathogenic DNA viruses (orthopoxvirus) are completely inactivated by a commercially available Roche MagNA Pure lysis/binding buffer as evaluated by subsequent cell culture. However, inactivation reagents are typically toxic and therefore problematic in cell culture. Using the relatively large orthopoxvirus, a method was developed in which virus is precipitated by high-speed centrifugation after inactivation but prior to application onto the target cells, thereby eliminating the cytotoxic effect of the lysis buffer. The results from quantitative PCR analysis indicate that the viral DNA from the completely inactivated virus particles, remain associated to macromolecules and aggregates. The use of inactivation buffers for bed-side inactivation of special patient samples taken for PCR diagnostics should be considered in cases where high containment would otherwise be required.