Viral Concentrates Research Articles

Concentration of Virus Particles from Environmental Water and Wastewater Samples Using Skimmed Milk Flocculation and Ultrafiltration.

Water and wastewater-based epidemiology have emerged as alternative methods to monitor and predict the course of outbreaks in communities.The recovery of microbial fractions, including viruses, bacteria, and microeukaryotes from wastewater and environmental water samplesis one of the challenging steps in these approaches. In this study, we focused on the recovery efficiency of sequential ultrafiltration and skimmed milk flocculation (SMF) methods using Armored RNA as a test virus, which is also used as a control by some other studies. Prefiltration with 0.45 µm and 0.2 µm membrane disc filters were applied to eliminate solid particles before ultrafiltration to prevent the clogging of ultrafiltration devices. Test samples, processed with the sequential ultrafiltration method, were centrifuged at two different speeds. An increased speed resulted in lower recovery and positivity rates of Armored RNA.On the other hand, SMF resulted in relatively consistent recovery and positivity rates of Armored RNA. Additional tests conducted with environmental water samples demonstrated the utility of SMF to concentrate other microbial fractions. The partitioning of viruses into solid particles might have an impact on the overall recovery rates, considering the prefiltration step applied before the ultrafiltration of wastewater samples. SMF with prefiltration performed better when applied to environmental water samplesdue to lower solid concentrations in the samples and thus lower partitioning rates to solids. In the present study, the idea of using a sequential ultrafiltration method arose from the necessity to decrease the final volume of the viral concentrates during the COVID-19 pandemic, when the supply of the commonly used ultrafiltration devices was limited, and there was a need for the development of alternative viral concentration methods.

Pilot study on wastewater surveillance of dengue virus RNA: Lessons, challenges, and implications for future research

Dengue virus (DENV) is an enveloped, single-stranded RNA virus that causes approximately 390 million infections, leading to 40,000 deaths annually. Due to the increasing trend of urbanization, water supply scarcity, and climate change, dengue is regarded as the “disease of the future,” requiring robust surveillance for the early detection of DENV infection. Since the virus is shed in urine and saliva and persists in wastewater at different temperatures, our study conducted wastewater surveillance as a novel approach to monitor dengue outbreaks in the Kathmandu Valley, Nepal. The viral concentrates (n = 34), which were previously collected and concentrated from municipal and hospital wastewater, and river water using the electronegative membrane-vortex method, were tested for DENV using quantitative reverse transcription polymerase chain reaction (RT-qPCR) and digital PCR (RT-dPCR). Pepper mild mottle virus, a process control and endogenous biomarker, was detected in all the samples with concentrations ranging from 8.0 to 10.0 log10 copies/L, whereas DENV was not detected in any sample using RT-dPCR and RT-qPCR. The undetected DENV in this study could be attributed to the collection of grab wastewater samples during a low relative prevalence of dengue infection in the region, insufficient sample volume processed, probable viral nucleic acid degradation due to storage of viral concentrate at -25 °C for a long period of time, or inefficiency of the primary concentration method used. This study highlights critical gaps in knowledge and provides recommendations for future implementation of wastewater surveillance of dengue outbreaks, especially in regions where dengue was recently introduced, clinical surveillance is limited, and wastewater surveillance for polio has been adopted.

First evidence of enterovirus A71 and echovirus 30 in Uruguay and genetic relationship with strains circulating in the South American region.

Human enteroviruses (EVs) comprise more than 100 types of coxsackievirus, echovirus, poliovirus and numbered enteroviruses, which are mainly transmitted by the faecal-oral route leading to diverse diseases such as aseptic meningitis, encephalitis, and acute flaccid paralysis, among others. Since enteroviruses are excreted in faeces, wastewater-based epidemiology approaches are useful to describe EV diversity in a community. In Uruguay, knowledge about enteroviruses is extremely limited. This study assessed the diversity of enteroviruses through Illumina next-generation sequencing of VP1-amplicons obtained by RT-PCR directly applied to viral concentrates of 84 wastewater samples collected in Uruguay during 2011–2012 and 2017–2018. Fifty out of the 84 samples were positive for enteroviruses. There were detected 27 different types belonging to Enterovirus A species (CVA2-A6, A10, A16, EV-A71, A90), Enterovirus B species (CVA9, B1-B5, E1, E6, E11, E14, E21, E30) and Enterovirus C species (CVA1, A13, A19, A22, A24, EV-C99). Enterovirus A71 (EV-A71) and echovirus 30 (E30) strains were studied more in depth through phylogenetic analysis, together with some strains previously detected by us in Argentina. Results unveiled that EV-A71 sub-genogroup C2 circulates in both countries at least since 2011–2012, and that the C1-like emerging variant recently entered in Argentina. We also confirmed the circulation of echovirus 30 genotypes E and F in Argentina, and reported the detection of genotype E in Uruguay. To the best of our knowledge this is the first report of the EV-A71 C1-like emerging variant in South-America, and the first report of EV-A71 and E30 in Uruguay.

SARS-CoV-2 in river water: Implications in low sanitation countries

Since the beginning of COVID-19 pandemic studies on viral shedding have reported that this virus is excreted in feces in most patients. High viral loads are found at the sewage pipeline or at the entrance of wastewater treatment plants from cities where the number of COVID-19 cases are significant. In Quito (Ecuador) as in many other cities worldwide, wastewater is directly discharged into natural waters. The aim of this study was to evaluate SARS-CoV-2 presence in urban streams from a low sanitation context. Three river locations along the urban rivers of Quito were sampled on the 5th of June during a peak of COVID-19 cases. River samples were evaluated for water quality parameters and afterwards, concentrated for viral analysis using skimmed milk flocculation method. The viral concentrates were quantified for SARS-CoV-2 (N1 and N2 target regions) and Human Adenovirus as a human viral indicator. The results showed that SARS-CoV-2 was detected for both target regions in all samples analyzed in a range of 2,91E+05 to 3,19E+06 GC/L for N1 and from 2,07E+05 to 2,22E+06 GC/L for N2. The high values detected in natural waters from a low sanitation region have several implications in health and ecology that should be further assessed.

Metaproteomics of marine viral concentrates reveals key viral populations and abundant periplasmic proteins in the oligotrophic deep chlorophyll maximum of the South China Sea.

Viral concentrates (VCs), containing bioinformative DNA and proteins, have been used to study viral diversity, viral metagenomics and virus-host interactions in natural ecosystems. Besides viruses, VCs also contain many noncellular biological components including diverse functional proteins. Here, we used a shotgun proteomic approach to characterize the proteins of VCs collected from the oligotrophic deep chlorophyll maximum (DCM) of the South China Sea. Proteins of viruses infecting picophytoplankton, that is, cyanobacteria and prasinophytes, and heterotrophic bacterioplankton, such as SAR11 and SAR116, dominated the viral proteome. Almost no proteins from RNA viruses or known gene transfer agents were detected, suggesting that they were not abundant at the sampling site. Remarkably, nonviral proteins made up about two thirds of VC proteins, including overwhelmingly abundant periplasmic transporters for nutrient acquisition and proteins for diverse cellular processes, that is, translation, energy metabolism and one carbon metabolism. Interestingly, three 56 kDa selenium-binding proteins putatively involved in peroxide reduction from gammaproteobacteria were abundant in the VCs, suggesting active removal of peroxide compounds at DCM. Our study demonstrated that metaproteomics provides a valuable avenue to explore the diversity and structure of the viral community and also the pivotal biological functions affiliated with microbes in the natural environment.

First evidence of the Hepatitis E virus in environmental waters in Colombia.

Hepatitis E virus (HEV) is one of the main causes of acute viral hepatitis of enteric transmission. HEV has been detected in environmental samples in several countries from Europe and Asia, constituting a risk factor for waterborne infection. In Colombia, HEV has been identified in samples obtained from patients as well as from swine, but no environmental studies have been carried out. To determine if HEV is present in environmental waters, samples from the main source of drinking water plant and of wastewater system of eight municipalities and two villages of Antioquia state (North West Colombia), were collected between December 2012 and April 2014. The HEV genome was detected by RT-PCR in 23.3% (7/30) of the samples from the main source of drinking water plants and in 16.7% (5/30) from sewage. Viral concentrates obtained from three positive sewage samples were used to inoculate HepG2 cell cultures that were followed for one month; however, the viral genome was not detected in any cell culture. This study demonstrates the circulation of HEV in both source of drinking water plants and wastewater in Antioquia state, Colombia. The presence of HEV in environmental waters could be a risk for waterborne transmission in this population. The findings of the present study, together with the evidence of HEV circulation in human and swine in Colombia, should be consider by public health authorities for the development of surveillance programs and the inclusion of HEV infection diagnosis in the guidelines of viral hepatitis in the country. This is the first report of HEV in environmental samples in Colombia and the second one in Latin America.

Tangential flow ultrafiltration for detection of white spot syndrome virus (WSSV) in shrimp pond water

Water represents the most important component in the white spot syndrome virus (WSSV) transmission pathway in aquaculture, yet there is very little information. Detection of viruses in water is a challenge, since their counts will often be too low to be detected by available methods such as polymerase chain reaction (PCR). In order to overcome this difficulty, viruses in water have to be concentrated from large volumes of water prior to detection. In this study, a total of 19 water samples from aquaculture ecosystem comprising 3 creeks, 10 shrimp culture ponds, 3 shrimp broodstock tanks and 2 larval rearing tanks of shrimp hatcheries and a sample from a hatchery effluent treatment tank were subjected to concentration of viruses by ultrafiltration (UF) using tangential flow filtration (TFF). Twenty to 100l of water from these sources was concentrated to a final volume of 100mL (200–1000 fold). The efficiency of recovery of WSSV by TFF ranged from 7.5 to 89.61%. WSSV could be successfully detected by PCR in the viral concentrates obtained from water samples of three shrimp culture ponds, one each of the shrimp broodstock tank, larval rearing tank, and the shrimp hatchery effluent treatment tank with WSSV copy numbers ranging from 6 to 157mL−1 by quantitative real time PCR. The ultrafiltration virus concentration technique enables efficient detection of shrimp viral pathogens in water from aquaculture facilities. It could be used as an important tool to understand the efficacy of biosecurity protocols adopted in the aquaculture facility and to carry out epidemiological investigations of aquatic viral pathogens.

Viral Metagenomics Reveal Blooms of Anelloviruses in the Respiratory Tract of Lung Transplant Recipients

Few studies have examined the lung virome in health and disease. Outcomes of lung transplantation are known to be influenced by several recognized respiratory viruses, but global understanding of the virome of the transplanted lung is incomplete. To define the DNA virome within the respiratory tract following lung transplantation we carried out metagenomic analysis of allograft bronchoalveolar lavage (BAL), and compared with healthy and HIV+ subjects. Viral concentrates were purified from BAL and analyzed by shotgun DNA sequencing. All of the BAL samples contained reads mapping to anelloviruses, with high proportions in lung transplant samples. Anellovirus populations in transplant recipients were complex, with multiple concurrent variants. Quantitative polymerase chain reaction quantification revealed that anellovirus sequences were 56-fold more abundant in BAL from lung transplant recipients compared with healthy controls or HIV+ subjects (p < 0.0001). Anellovirus sequences were also more abundant in upper respiratory tract specimens from lung transplant recipients than controls (p = 0.006). Comparison to metagenomic data on bacterial populations showed that high anellovirus loads correlated with dysbiotic bacterial communities in allograft BAL (p = 0.008). Thus the respiratory tracts of lung transplant recipients contain high levels and complex populations of anelloviruses, warranting studies of anellovirus lung infection and transplant outcome.

Influence of Hot Spring Phages on Community Carbon Metabolism: Win, Lose or Draw?

Abundant virus-like particles were concentrated from large-volume samples from two hot springs. Both addition of viral concentrates and addition of samples induced by addition of mitomycin-C changed patterns of carbon source utilization by hot spring microbial communities. Specific effects of the two treatments depended upon both temperature and incubation period. Increased metabolic capability with greater exposure to free phages, consistent with the view that phages are major lateral transporters of metabolic genes, was observed most clearly in microbes incubated at a temperature lower than that encountered in situ. On the other hand, decreases in the diversity of utilizable C sources upon exposure to phages may have been due to lytic activity in which susceptible bacterial populations were differentially reduced by infective viruses, consistent with the “killing the winner” hypothesis. Treatment of cultures with MC-treated culture extracts, assumed to increase exposure to excised prophages, resulted in higher average metabolic rates after 18 h, but lower rates after 48 h of incubation. With incubation at in situ temperature, this same treatment led to an initial increase in the number of readily utilized C sources, followed by a decrease in community metabolic diversity relative to controls in samples from both hot springs. Thus, treatments designed to increase the interaction between hot spring microbes and either free or newly-excised phages had observable time- and temperature-dependent effects on community metabolism, demonstrating an important, yet complex, ecological role for phages in hot spring waters.

Efficient purification and concentration of viruses from a large body of high turbidity seawater

Marine viruses are the most abundant entities in the ocean and play crucial roles in the marine ecological system. However, understanding of viral diversity on large scale depends on efficient and reliable viral purification and concentration techniques. Here, we report on developing an efficient method to purify and concentrate viruses from large body of high turbidity seawater. The developed method characterizes with high viral recovery efficiency, high concentration factor, high viral particle densities and high-throughput, and is reliable for viral concentration from high turbidity seawater. Recovered viral particles were used directly for subsequent analysis by epifluorescence microscopy, transmission electron microscopy and metagenomic sequencing. Three points are essential for this method:•The sampled seawater (>150L) was initially divided into two parts, water fraction and settled matter fraction, after natural sedimentation.•Both viruses in the water fraction concentrated by tangential flow filtration (TFF) and viruses isolated from the settled matter fraction were considered as the whole viral community in high turbidity seawater.•The viral concentrates were re-concentrated by using centrifugal filter device in order to obtain high density of viral particles.

Unique viral capsid assembly protein gene (g20) of cyanophages in the floodwater of a Japanese paddy field

In order to evaluate the genetic diversity of cyanophage communities of rice fields, viral capsid assembly protein gene (g20) was amplified with primers CPS1 and CPS8. The DNA was extracted three times from viral concentrates obtained from floodwater samples collected in each of four different plots (no fertilizer; P and K chemical fertilizers; N, P, and K chemical fertilizers; and chemical fertilizers with compost). Denaturing gradient gel electrophoresis (DGGE) gave different g20 clones. The sequencing of DGGE bands revealed that the g20 genes of the floodwater were divergent and that the majority of clones formed several unique groups. However, they were more closely related to g20 sequences from freshwaters than to those from marine waters, suggesting that g20 genes in terrestrial aquatic environments are different from those in marine environments.

Novel cyanophage photosynthetic gene psbA in the floodwater of  a Japanese rice field

The gene psbA, encoding the D1 protein involved in photosynthesis, was recently found in a number of cultured cyanophages infecting marine Synechococcus and Prochlorococcus and in environmental samples from marine and freshwaters. In this study, viral concentrates were prepared by sampling the floodwaters from each of four plots in a Japanese rice field: (1) no fertilizer; (2) P and K chemical fertilizers; (3) N, P and K chemical fertilizers; and (4) chemical fertilizers with compost. Fragments of the cyanophage psbA gene were amplified by PCR from DNA in the viral concentrates, with primers psbA-F and psbA-R. Double denaturing gradient gel electrophoresis was conducted to obtain different psbA clones. Phylogenetic analyses indicated that the majority of the psbA sequences in the floodwater formed two unique groups, with their sequences being more closely related to those from freshwater samples than the sequences obtained from marine waters, suggesting that psbA genes in terrestrial aquatic environments are different from those in marine environments.

Movement of viruses between biomes.

Viruses are abundant in all known ecosystems. In the present study, we tested the possibility that viruses from one biome can successfully propagate in another. Viral concentrates were prepared from different near-shore marine sites, lake water, marine sediments, and soil. The concentrates were added to microcosms containing dissolved organic matter as a food source (after filtration to allow 100-kDa particles to pass through) and a 3% (vol/vol) microbial inoculum from a marine water sample (after filtration through a 0.45-microm-pore-size filter). Virus-like particle abundances were then monitored using direct counting. Viral populations from lake water, marine sediments, and soil were able to replicate when they were incubated with the marine microbes, showing that viruses can move between different ecosystems and propagate. These results imply that viruses can laterally transfer DNA between microbes in different biomes.

The effect of viral concentrate addition on the respiration rate of Chaetoceros gracilis cultures and microplankton from a shallow bay (Coliumo, Chile)

Experiments were conducted to test the possible effects of viral concentrate addition on the respiratioin rates of both Chaetoceros gracilis and a natural microplankton community (<200 μm) from a shallow bay located in central-south Chile (Coliumo Bay, 36°32'S, 72°57'@). Each experiment was started by adding 2 ml of viral concentrate to a C. Gracilis culture, microplankton community or bacterioplankton community (nominal size 0.2-1.0 μm) contained in a 125 ml borosilicate bottle. The incubations lasted 24 h at in situ temperatures and included a minimum of five replicates and five controls. Respiration was measured as oxygen consumption using a semi-automatic photometric Winkler method. Samples were taken from the experimental bottles to assess chlorophyll a, ATP concentration and bacterioplankton abundance throughout the incubation period. Our results show that the addition of viral concentrates can affect the respiration rates of both natural microplankton communities and C. gracilis cultures. When subjected to a viral concentrate addition, the respiration rates of the natural microplankton community decreased by 12 -50% or increased by 29%, depending on the experiment, whereas bacterioplankton respiration rates increased by 92% and decreased by 78%, and C. gracilis rates increased by ∼4% and decreased by ∼ 7%.

Modified Concentration Method for the Detection of Enteric Viruses on Fruits and Vegetables by Reverse Transcriptase-Polymerase Chain Reaction or Cell Culture

Fruits and vegetables may act as a vehicle of human enteric virus if they are irrigated with sewage-contaminated water or prepared by infected food handlers. An elution-concentration method was modified to efficiently detect, by reverse transcriptase-polymerase chain reaction (RT-PCR) or by cell culture, contamination by poliovirus, hepatitis A virus (HAV), and Norwalk-like virus (NLV) of various fresh and frozen berries and fresh vegetables. The protocol included washing the fruit or vegetable surface with 100mM Tris-HCl, 50mM glycine, and 3% beef extract, pH 9.5 buffer, which favors viral elution from acid-releasing berries, supplemented with 50mM MgCl2 to reduce the decrease in viral infectivity during the process. The viral concentration method was based on polyethylene glycol precipitation. Copurified RT-PCR inhibitors and cytotoxic compounds were removed from viral concentrates by chloroform-butanol extraction. Viruses from 100 g of vegetal products could be recovered in volumes of 3 to 5 ml. Viral RNAs were isolated by a spin column method before molecular detection or concentrates were filtered (0.22-μm porosity) and inoculated on cell culture for infectious virus detection. About 15% of infectious poliovirus and 20% of infectious HAV were recovered from frozen raspberry surfaces. The percentage of viral RNA recovery was estimated by RT-PCR to be about 13% for NLV, 17% for HAV, and 45 to 100% for poliovirus. By this method, poliovirus and HAV RNA were detected on products inoculated with a titer of about 5 × 101 50% tissue culture infectious dose per 100 g. NLV RNA was detected at an initial inoculum of 1.2 × 103 RT-PCR amplifiable units. This method would be useful for the viral analysis of fruits or vegetables during an epidemiological investigation of foodborne diseases.

Use of Retroviral Vectors to Introduce Antisense Sequences into Embryonic Chick Brain

Recombinant retroviral vectors have been used as tracers to analyze cell lineage and migration in several parts of the nervous system. In such studies, neural progenitor cells are infected with a vector containing a reporter gene such as Escherichia coli β-galactosidase ( lacZ) or alkaline phosphatase; the gene product is later detected histochemically in that cells clonal progeny. We have now extended this cell-marking method to introduce antisense sequences along with lacZ, in order to determine cell-autonomous effects of attenuating specific neural genes in vivo. To test the strategy, we used the chicken β 1-integrin gene. Antisense sequences covering the translation Initiation sequence were fused to lacZ in a retroviral vector, and virions were produced in helper cells. Tests on cultured cells showed that levels of cell surface β 1-integrin were significantly and selectively reduced following transfection or infection with antisense vectors. Viral concentrates were then injected into midbrain vesicles of chick embryos while neuronal progenitors were dividing. At later stages, tecta were fixed and stained for lacZ with X-gal, and clones of lacZ-positive cells were analyzed. Cells in antisense-bearing clones accumulated in the ventricular zone and failed to migrate along radial glia into the forming tectal plate. At late stages, few antisense-containing cells could be found, suggesting that attenuation of integrin expression was lethal. Thus, tectal cells appear to require β 1-integrins for migration and survival. We suggest that retrovirus-mediated transfer of antisense sequences may be generally useful for determining cell-autonomous functions of selected molecules in developing neural tissues. Our results also show that antisense RNA can be effective as part of a larger transcript, thus simplifying its use in vectors that contain lacZ.

Presence of human immunodeficiency virus nucleic acids in wastewater and their detection by polymerase chain reaction.

The human immunodeficiency virus type 1 (HIV-1) released by infected individuals or present in human and hospital wastes can potentially cause contamination problems. The presence of HIV-1 was investigated in 16 environmental samples, including raw wastewater, sludge, final effluent, soil, and pond water, collected from different locations. A method was developed to extract total nucleic acids in intact form directly from the raw samples or from the viral concentrates of the raw samples. The isolated nucleic acids were analyzed for the presence of HIV-1 by using in vitro amplification of the target sequences by the polymerase chain reaction (PCR) method. HIV-1-specific proviral DNA and viral RNA were detected in the extracted nucleic acids obtained from three wastewater samples by this method. The specificity of the PCR-amplified products was determined by Southern blot hybridization with an HIV-1-specific oligonucleotide probe, SK19. The isolated nucleic acids from wastewater samples were also screened for the presence of poliovirus type 1, representing a commonly found enteric virus, and simian immunodeficiency virus, representing, presumably, rare viruses. While poliovirus type 1 viral RNA was found in all of the wastewater samples, none of the samples yielded a simian immunodeficiency virus-specific product. No PCR-amplified product was yielded when wastewater samples were directly used for the detection of HIV-1 and poliovirus type 1. The wastewater constituents appeared to be inhibitory to the enzymes reverse transcriptase and DNA polymerase.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-HCV and recipients: situation in hemophiliacs in 1991

Sera from 273 French haemophiliacs who had received non viral inactivated concentrates, were tested for antibodies to HCV by first and second generation assays. Antibodies to HCV were detected in 66% of the sera by the first generation assays (anti-C 100-3) reaching 100% by the second generation assays. None of the 53 patients only exposed to solvent-detergent treated Factor VIII or IX concentrates had HCV seroconversion. HCV core protein antibody was always detectable often as a single antibody in seropositive hemophilic patients.

Cell-free transmission of mouse neuroblastoma.

AFTER the description of the cell-free transmission of neuroblastoma C1300, a transplantable tumour in strain A/J mice, by Prasad et al.1, we made ten attempts to reproduce their results, first using their extraction procedure and then by using the viral concentration procedure of Moloney2, modified by Huebner et al.3. We inoculated twenty-eight prenatal, forty-seven newborn, and sixteen 4-week-old A/J mice but failed to obtain positive results, even after 100 days postinoculation. Because we used newborn mice that are usually more susceptible to oncogenic viruses than the older mice used by Prasad et al., and viral concentrates rather than filtrates, we expected to increase our chances of transmission.

The zonal centrifuge applied to the purification of a low‐yield virus in human leukemia cells

AbstractThe herpes‐type virus found in certain cell cultures derived from Burkitt's lymphoma, other human leukemias, and normal human leukocytes, was concentrated and partially purified by large‐volume density gradient centrifugation using zonal centrifuge systems. Using the Jiyoye (P‐3) cell line as a model, rate‐zonal runs on disrupted cell suspensions in sucrose gradients yielded concentrates with high virus particle counts when 10–15 ml of packed cells were processed per liter of gradient. Isolation and removal of cell nuclei or fluorocarbon treatment of cell sonicates permitted virus recovery from larger volumes of cells per experiment. Zonal centrifugation of concentrated cell‐free spent media from highly infected cell cultures yielded more purified virus than obtained from cells. Viral concentrates were prepared with particle counts of 1010–1011/ml and total protein concentrations of 0.2–0.5 mg/ml. Subsequent isopyenie‐zonal centrifugation of the various high‐count virus fractions from the zonal centrifuge showed a heterogeneity in buoyant virus density ranging from 1.18 to 1.27 in potassium tart rate. The spread in virus density was attributed to the different morphological forms of the virus observed by electron microscopy.