Klassevirus 1, a previously undescribed member of the family Picornaviridae, is globally widespread
© Holtz et al; licensee BioMed Central Ltd. 2009
Received: 04 June 2009
Accepted: 24 June 2009
Published: 24 June 2009
Diarrhea is the third leading infectious cause of death worldwide and is estimated to be responsible for approximately 2 million deaths a year. While many infectious causes of diarrhea have been established, approximately 40% of all diarrhea cases are of unknown etiology. In an effort to identify novel viruses that may be causal agents of diarrhea, we used high throughput mass sequencing to analyze stool samples collected from patients with acute diarrhea.
Sequences with limited similarity to known picornaviruses were detected in a stool sample collected in Australia from a child with acute diarrhea. Using a combination of mass sequencing, RT-PCR, 5' RACE and 3' RACE, a 6383 bp fragment of the viral genome was sequenced. Phylogenetic analysis demonstrated that this virus was highly divergent from, but most closely related to, members of the genus Kobuvirus. We have tentatively named this novel virus klassevirus 1. We also detected klassevirus 1 by RT-PCR in a diarrhea specimen collected from a patient in St. Louis, United States as well as in untreated sewage collected in Barcelona, Spain.
Klassevirus 1 is a previously undescribed picornavirus that is globally widespread and present on at least three continents. Further investigations to determine whether klassevirus 1 is a human pathogen are needed.
The impact of diarrhea is primarily felt in the developing world, where approximately 2 million deaths result from diarrhea annually [1–3]. In developed countries, where diarrhea related mortality is relatively rare, there is still nonetheless a tremendous disease burden. For example, in the United States, approximately 9% of all hospitalizations for children under age 5 years are due to diarrhea episodes . While rotaviruses, caliciviruses, adenoviruses, and astroviruses are responsible for the greatest proportion of cases [5–8], approximately 40% of diarrhea cases are of unknown etiology [9–11].
Many picornaviruses can be detected in human stool such as enteroviruses, polio, Aichi virus, and cardioviruses [12–15]. Some of these viruses, such as Aichi virus, are associated with diarrheal disease  while others such as polio are shed fecally, but manifest pathogenicity in other organ systems. Picornaviruses are non-enveloped viruses with a single stranded positive-sense RNA genome that encodes a single polyprotein . The picornavirus family currently consists of 14 proposed genera http://www.picornaviridae.com associated with a diverse range of diseases. Viruses in six of these genera potentially infect humans (Enterovirus, Hepatovirus, Parechovirus, Kobuvirus, Cosavirus, and Cardiovirus). With the advent of culture independent molecular methods, many diverse new members of the picornavirus family have been identified in recent years. These include novel cardioviruses [14–17], rhinoviruses [18–20], parechoviruses [21, 22] and the novel genus of cosaviruses [23, 24]. These studies have demonstrated that significant viral diversity exists in the human gut that remains unexplored.
We have previously described a mass sequencing strategy based on high throughput Sanger sequencing to analyze human stool for previously undescribed viruses . In this study, we used a similar strategy but incorporated a next-generation pyrosequencing platform (Roche Genome Sequencer) in place of traditional Sanger sequencing. This resulted in the identification of a highly divergent picornavirus in a stool sample collected in 1984 from a child in Australia with acute diarrhea. Sequencing and phylogenetic analysis demonstrated that this virus is a novel member of the family Picornaviridae. We propose that this virus be named klassevirus 1 (k obu-l ike virus a ssociated with s tool and se wage).
Identification and sequencing of klassevirus 1
Extracted nucleic acid from a stool specimen collected in 1984 from a child with acute diarrhea was subjected to high throughput mass sequencing using 454 pyroseqencing technology. From the resulting reads, two sequences were identified by BLAST that had only limited sequence identity to known viruses. One was a 217 bp fragment that upon translation to amino acid sequence had 42% identity to its closest relative, Aichi virus (2B/2C region). The second sequence read of 443 bp had 40% amino acid identity to the VP0 region of Aichi virus. From these two initial sequences, a 6383 bp contig (klasse-mel1) was generated by RT-PCR and multiple 3' and 5' random amplification of cDNA ends (RACE) reactions. The 5' end of this contig aligned to the predicted VP0 protein at the N-terminus of the polyprotein and extended past the predicted 3D protein at the C-terminus of the polyprotein to the poly-A tail. The initial assembly was confirmed by sequencing multiple overlapping RT-products spanning the length of the contig to give 3.3X coverage. We were not able to extend the contig further in the 5' direction despite performing multiple 5' RACE reactions using different primers with multiple high temperature (70°C) reverse transcriptases (rTth [Applied Biosystems] and Thermoscript [Invitrogen]).
Phylogenetic analysis of klassevirus 1
Epidemiological survey for klassevirus 1
Pair-wise amino acid identities between three klassevirus 1 strains using partial sequence fragments.
To further compare the divergence between the three positive samples of klassevirus 1, RT-PCR was performed using primers that target the polymerase region (3D) of the genome and the VP0/VP3 region. Approximately one kb of additional sequence was generated from klasse-bar1 and klasse-stl1 in both of these regions (see methods). Pair-wise amino acid identities ranged from 85%–97%, with the greatest degree of sequence conservation in the 3D region (Table 1).
In this study, we identified a previously undescribed picornavirus present in stool and sewage. Phylogenetic analysis demonstrated that this virus is most closely related to other picornaviruses in the genus Kobuvirus. Based on the criteria established by the picornavirus study group, members of a genus should share > 40%, > 40% and > 50% amino acid identity in P1, P2 and P3 genome regions respectively . Klassevirus 1 shared only 43% amino acid identity in the P3 region and 33% amino acid identity in the P2 region to its closest relative, Aichi virus. Given these observations, and using strictly the percent identity definitions, klassevirus 1 may represent the first member of new picornavirus genus. However, we note that at all loci, bootstrap analysis suggests that klassevirus 1 diverged from an ancestor common to all of the known kobuviruses. Thus the formal classification of klassevirus 1 at the genus level is currently uncertain and subject to further discussion per the ICTV.
Subsequent screening by RT-PCR using primers targeting the 2C region of the genome established that klassevirus 1-like sequences were present not only in Australia, but also in North America and Europe. The presence of klassevirus 1 in the United States was determined by the traditional strategy of screening of individual stool samples. In addition, we also examined raw sewage collected in Barcelona to see if we could detect klassevirus 1. Sewage represents a pooled meta-sample of literally thousands of individual specimens. Known enteric viruses such as adenoviruses [28, 29], noroviruses  astroviruses , and hepatitis A  have frequently been tested for and detected in sewage by PCR and RT-PCR. We reasoned that detection of klassevirus 1 in raw sewage would serve as a proxy for its presence in human stool in the population that generated the sewage. Since the exact history of the sewage is poorly defined, it is possible that other waste products, such as animal feces could contribute to the raw sewage meta-genome. Nonetheless, we propose that raw sewage screening from a diversity of sites can serve to rapidly define the geographic distribution of a given virus. The detection of klassevirus 1 in stool and sewage from Melbourne, Barcelona and St. Louis, demonstrates that klassevirus 1 is globally distributed. Moreover, since both the Barcelona sewage and St. Louis stool specimens were collected in 2008, we conclude that klassevirus 1 is currently circulating in the human population.
Whether klassevirus 1 represents a true human pathogen remains to be determined. It is possible that klassevirus 1 is a human pathogen that causes gastroenteritis. It is also possible that klassevirus 1 injures other organs but is excreted through the intestinal tract like poliovirus. Another possibility is that klassevirus 1 is a human commensal virus. Alternatively, klassevirus 1 could represent a non-human virus acquired from dietary exposure. Further investigations are needed to determine if klassevirus 1 is a causal agent of human disease(s). To begin addressing this question, epidemiologic studies including case-control and seroprevalence analyses are needed.
Materials and methods
Primary Stool Specimen
This stool was collected in 1984 from a 38 month old child presenting to the emergency department of the Royal Children's Hospital, Melbourne, Australia with acute diarrhea and stored at -80°C. Previous testing of this diarrhea specimen for known enteric pathogens using routine enzyme immunoassays (EIA) and culture assays for rotaviruses, adenoviruses, and common bacterial and parasitic pathogens was negative . Additionally, RT-PCR assays for caliciviruses and astroviruses were also negative [6, 33].
Sample preparation for 454 sequencing
120 mg of frozen stool was chipped and then resuspended in 6 volumes of PBS . The sample was centrifuged to pellet particulate matter and the supernatant was then passed through a 0.45 μm filter. Total nucleic acid was isolated from 100 μL primary stool filtrate using QiAmp DNA extraction kit (Qiagen) according to manufacturer's instructions. Total nucleic acid was randomly amplified using the Round AB protocol as previously described . This was then pyrosequenced on a Roche FLX Genome sequencer (Roche) according to manufacturer's protocol. To eliminate sequence redundancy in each library sequences were clustered using BLASTCLUST from the 2.2.17 version of NCBI BLAST. Sequences were clustered based on 98% identity over 98% sequence length and the longest sequence from each cluster was chosen as the representative sequence of the cluster. Unique sequences were filtered for repetitive sequences and then compared with the GenBank nr database by BLASTN and TBLASTX.
Sequencing of klassevirus 1
For sequencing experiments, the stool filtrate was proteinase K treated prior to RNA extraction. RNA was isolated from primary stool filtrate using RNA-Bee (Tel-Test, Inc.) according to manufacturer's instructions. RT-PCR and 3'RACE reactions were performed using SuperScript III and Platinum Taq (Invitrogen One-Step RT-PCR). For 5'RACE reactions cDNA was generated with Themoscript (Invitrogen) and amplified with Accuprime Taq (Invitrogen). Amplicons were either cloned into pCR4 (Invitrogen) or sequenced directly.
Protein sequences associated with the following reference virus genomes were obtained from GenBank: Equine Rhinitis A virus (NP_653075.1), Foot-and-mouth-type-O (NP_658990.1), Equine Rhinitis B virus (NP_653077.1), Theiler murine encephalomyelitis (AAA47929.1), Mengo virus (AAA46547.1), Encephalomyocarditis virus (CAA60776.1), Seneca valley virus (DQ641257), Aichi virus (NP_047200.1), Porcine teschovirus (NP_653143.1), Human Cosavirus E-1 (FJ555055.1), Hepatitis A Virus (M14707), Bovine kobuvirus (NP_740257.1), Porcine kobuvirus (YP_002456506.1), Human coxsackievirus A2 (AAR38840.1), Porcine enterovirus A (NC_003987), Human poliovirus 2 (M12197), Avian encephalomyelitis virus (NC_003990), Duck hepatitis virus 1 (ABI23434), Seal picornavirus type 1 (NC_009891), Human Cosavirus A1 (FJ438902), and Human parechovirus 1 (AAA72291.1). Multiple sequence alignments were performed using ClustalX (1.83). The amino acid alignments generated by ClustalX were input into PAUP , and maximum parsimony analysis was performed using the default settings with 1,000 replicates.
Epidemiological survey for klassevirus 1
Stool samples were collected from children under the age of 5 who were admitted to the Royal Children's Hospital, Melbourne, Victoria, Australia with acute diarrhea between 1978 and 1999. For a portion of these samples (70), RNA was extracted in the same manner as the primary sample. For the remaining specimens (73), chips of frozen fecal specimens (~30–150 mg) were resuspended in 6 volumes of PBS. Total nucleic acid was extracted from 200 μL of each stool suspension using a MagnaPure LC instrument (Roche). 200 μL of water was used to elute the total nucleic acid from each sample.
St. Louis Cohort
Leftover material from 340 stool specimens that were routinely submitted to the St. Louis Children's Hospital Lab for bacterial culture were collected from January 2008–July 2008. For these specimens total nucleic acid was extracted as described above. This study was approved by the Human Research Protection Office of Washington University.
One 10 L-sample of raw sewage was collected in an urban wastewater treatment plant in the area of Barcelona, Spain. The sample was collected in a sterile container and stored for up to 2 hours at 4°C before being processed. The viruses present in the sample were concentrated in 30 mL of phosphate buffer by organic flocculation based on the procedure previously described by Calgua et al., 2008 . A second concentration step with elution of the viral particles was performed. Briefly, 10 mL of the viral concentrate were eluted with 40 mL of 0.25 M glycine buffer (pH 9,5) at 4°C, suspended solids were separated by low speed centrifugation at 7500 × g for 30 min at 4°C and the viruses present in the supernatant were finally concentrated in 1 mL of PBS by ultracentrifugation at 87500 × g for 1 h at 4°C. This was then DNase treated, and then total nucleic acid was extracted.
RT-PCR for detection of klassevirus 1
Primers expected to generate a 345 bp product were designed to the 2C region of klassevirus 1 (LG0098: 5'-CGTCAGGGTGTTCGTGATTA-3' and LG0093: 5'-AGAGAGAGCTGTGGAGTAATTAGTA-3'). RT-PCR reactions were performed using Qiagen one-step kit under the following conditions: 30 min RT step, 94°C hold for 10 min, followed by 40 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 60 s. In order to further compare strain divergence, primers expected to produce amplicons of 1001 bp and 1025 bp based on the klasse-mel1 sequence were designed targeting the 3D and VP0/VP3 regions, respectively: (LG0118: 5'-ATGGCAACCCTGTCCCTGAG-3' and LG0117 5'-GGAAACCCAACCACGCTGTA-3') and (LG0119: 5'-GCTAACTCTAATGCTGCCACC-3' and LG0136: 5'-GCTAGGTCAGTGGAAGGATCA-3'). These RT-PCR reactions were performed using the Invitrogen One-Step RT-PCR kit with the following conditions: 30 min RT step at 60°C, 94°C hold for 2 min, followed by 40 cycles of 94°C for 15 s, 56°C for 30 s, 68°C for 90 s. Whenever possible, amplicons were cloned into pCR4 (Invitrogen) and sequenced using standard Sanger sequencing technology. In some instances, PCR products were directly sequenced and only high quality sequence from those samples were included in analysis. All klassevirus 1 sequences have been deposited in Genbank (GQ253930-GQ253936).
This research was supported in part by the National Institutes of Health under Ruth L. Kirschstein National Research Service Award (5 T32 DK077653) from the NIDDK and by National Institutes of Health grant U54AI057160 to the Midwest Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research. DW holds an Investigator in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund. CK is supported by an NHMRC RD Wright Research Fellowship (ID 334364). We would like to thank Drs. Gregory Storch and Binh-Minh Le for their help in the accrual and processing of the St. Louis stool specimens and Dr. Joseph Derisi and his laboratory at UC San Francisco for sharing independently derived data prior to publication.
- World Health Report 2004 World Health Organization, Geneva, Switzerland; 2004.
- Kosek M, Bern C, Guerrant RL: The global burden of diarrhoeal disease, as estimated from studies published between 1992 and 2000. Bull World Health Organ 2003, 81: 197-204.PubMed CentralPubMedGoogle Scholar
- O'Ryan M, Prado V, Pickering LK: A millennium update on pediatric diarrheal illness in the developing world. Semin Pediatr Infect Dis 2005, 16: 125-136. 10.1053/j.spid.2005.12.008View ArticlePubMedGoogle Scholar
- Chabra A, Chavez GF, Taylor D: Hospital use by pediatric patients: implications for change. Am J Prev Med 1997, 13: 30-37.PubMedGoogle Scholar
- Clark B, McKendrick M: A review of viral gastroenteritis. Curr Opin Infect Dis 2004, 17: 461-469. 10.1097/00001432-200410000-00011View ArticlePubMedGoogle Scholar
- Kirkwood CD, Clark R, Bogdanovic-Sakran N, Bishop RF: A 5-year study of the prevalence and genetic diversity of human caliciviruses associated with sporadic cases of acute gastroenteritis in young children admitted to hospital in Melbourne, Australia (1998–2002). J Med Virol 2005, 77: 96-101. 10.1002/jmv.20419View ArticlePubMedGoogle Scholar
- Klein EJ, Boster DR, Stapp JR, Wells JG, Qin X, Clausen CR, Swerdlow DL, Braden CR, Tarr PI: Diarrhea etiology in a Children's Hospital Emergency Department: a prospective cohort study. Clin Infect Dis 2006, 43: 807-813. 10.1086/507335View ArticlePubMedGoogle Scholar
- Wilhelmi I, Roman E, Sanchez-Fauquier A: Viruses causing gastroenteritis. Clin Microbiol Infect 2003, 9: 247-262. 10.1046/j.1469-0691.2003.00560.xView ArticlePubMedGoogle Scholar
- Denno DM, Klein EJ, Young VB, Fox JG, Wang D, Tarr PI: Explaining unexplained diarrhea and associating risks and infections. Anim Health Res Rev 2007, 8: 69-80. 10.1017/S1466252307001302View ArticlePubMedGoogle Scholar
- Kapikian AZ: Viral gastroenteritis. Jama 1993, 269: 627-630. 10.1001/jama.269.5.627View ArticlePubMedGoogle Scholar
- Chikhi-Brachet R, Bon F, Toubiana L, Pothier P, Nicolas JC, Flahault A, Kohli E: Virus diversity in a winter epidemic of acute diarrhea in France. J Clin Microbiol 2002, 40: 4266-4272. 10.1128/JCM.40.11.4266-4272.2002PubMed CentralView ArticlePubMedGoogle Scholar
- Racaniello VR: Picornaviridae: The Viruses and Their Replication. In Fields Virology. Volume 1. 5th edition. Edited by: Howley DMKaPM. Philadelphia: Lippincott Williams & Wilkins; 2007:795-838.Google Scholar
- Yamashita T, Kobayashi S, Sakae K, Nakata S, Chiba S, Ishihara Y, Isomura S: Isolation of cytopathic small round viruses with BS-C-1 cells from patients with gastroenteritis. J Infect Dis 1991, 164: 954-957.View ArticlePubMedGoogle Scholar
- Jones MS, Lukashov VV, Ganac RD, Schnurr DP: Discovery of a novel human picornavirus in a stool sample from a pediatric patient presenting with fever of unknown origin. J Clin Microbiol 2007, 45: 2144-2150. 10.1128/JCM.00174-07PubMed CentralView ArticlePubMedGoogle Scholar
- Chiu CY, Greninger AL, Kanada K, Kwok T, Fischer KF, Runckel C, Louie JK, Glaser CA, Yagi S, Schnurr DP, Haggerty TD, Parsonnet J, Ganem D, DeRisi JL: Identification of cardioviruses related to Theiler's murine encephalomyelitis virus in human infections. Proc Natl Acad Sci USA 2008, 105: 14124-14129. 10.1073/pnas.0805968105PubMed CentralView ArticlePubMedGoogle Scholar
- Abed Y, Boivin G: New Saffold cardioviruses in 3 children, Canada. Emerg Infect Dis 2008, 14: 834-836. 10.3201/eid1405.071459PubMed CentralView ArticlePubMedGoogle Scholar
- Drexler JF, Luna LK, Stöcker A, Almeida PS, Ribeiro TC, Petersen N, Herzog P, Pedroso C, Huppertz HI, Ribeiro Hda C Jr, Baumgarte S, Drosten C: Circulation of 3 lineages of a novel Saffold cardiovirus in humans. Emerg Infect Dis 2008, 14: 1398-1405. 10.3201/eid1409.080570PubMed CentralView ArticlePubMedGoogle Scholar
- Lamson D, Renwick N, Kapoor V, Liu Z, Palacios G, Ju J, Dean A, St George K, Briese T, Lipkin WI: MassTag polymerase-chain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness in New York State during 2004–2005. J Infect Dis 2006, 194: 1398-1402. 10.1086/508551View ArticlePubMedGoogle Scholar
- McErlean P, Shackelton LA, Lambert SB, Nissen MD, Sloots TP, Mackay IM: Characterisation of a newly identified human rhinovirus, HRV-QPM, discovered in infants with bronchiolitis. J Clin Virol 2007, 39: 67-75. 10.1016/j.jcv.2007.03.012View ArticlePubMedGoogle Scholar
- Kistler A, Avila PC, Rouskin S, Wang D, Ward T, Yagi S, Schnurr D, Ganem D, DeRisi JL, Boushey HA: Pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity. J Infect Dis 2007, 196: 817-825. 10.1086/520816View ArticlePubMedGoogle Scholar
- Drexler JF, Grywna K, Stocker A, Almeida PS, Medrado-Ribeiro TC, Eschbach-Bludau M, Petersen N, da Costa-Ribeiro H Jr, Drosten C: Novel human parechovirus from Brazil. Emerg Infect Dis 2009, 15: 310-313. 10.3201/eid1502.081028PubMed CentralView ArticlePubMedGoogle Scholar
- Li L, Victoria J, Kapoor A, Naeem A, Shaukat S, Sharif S, Alam MM, Angez M, Zaidi SZ, Delwart E: Genomic characterization of novel human parechovirus type. Emerg Infect Dis 2009, 15: 288-291. 10.3201/eid1502.080341PubMed CentralView ArticlePubMedGoogle Scholar
- Kapoor A, Victoria J, Simmonds P, Slikas E, Chieochansin T, Naeem A, Shaukat S, Sharif S, Alam MM, Angez M, Wang C, Shafer RW, Zaidi S, Delwart E: A highly prevalent and genetically diversified Picornaviridae genus in South Asian children. Proc Natl Acad Sci USA 2008, 105: 20482-20487. 10.1073/pnas.0807979105PubMed CentralView ArticlePubMedGoogle Scholar
- Holtz LR, Finkbeiner SR, Kirkwood CD, Wang D: Identification of a novel picornavirus related to cosaviruses in a child with acute diarrhea. Virol J 2008, 5: 159. 10.1186/1743-422X-5-159PubMed CentralView ArticlePubMedGoogle Scholar
- Finkbeiner SR, Allred AF, Tarr PI, Klein EJ, Kirkwood CD, Wang D: Metagenomic analysis of human diarrhea: viral detection and discovery. PLoS Pathog 2008, 4: e1000011. 10.1371/journal.ppat.1000011PubMed CentralView ArticlePubMedGoogle Scholar
- Finn RD, Mistry J, Schuster-Böckler B, Griffiths-Jones S, Hollich V, Lassmann T, Moxon S, Marshall M, Khanna A, Durbin R, Eddy SR, Sonnhammer EL, Bateman A: Pfam: clans, web tools and services. Nucleic Acids Res 2006, 34: D247-251. 10.1093/nar/gkj149PubMed CentralView ArticlePubMedGoogle Scholar
- Stanway G, Brown F, Christian P, Hovi T, Hyypiä T, King AMQ, Knowles NJ, Lemon SM, Minor PD, Pallansch MA, Palmenberg AC, Skern T: Family Picornaviridae. In Virus Taxonomy Eighth Report of the International Committee on Taxonomy of Viruses. Edited by: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA. London: Elsevier/Academic Press; 2005:757-778.Google Scholar
- Puig M, Jofre J, Lucena F, Allard A, Wadell G, Girones R: Detection of adenoviruses and enteroviruses in polluted waters by nested PCR amplification. Appl Environ Microbiol 1994, 60: 2963-2970.PubMed CentralPubMedGoogle Scholar
- Pina S, Puig M, Lucena F, Jofre J, Girones R: Viral pollution in the environment and in shellfish: human adenovirus detection by PCR as an index of human viruses. Appl Environ Microbiol 1998, 64: 3376-3382.PubMed CentralPubMedGoogle Scholar
- Lodder WJ, Vinje J, Heide R, de Roda Husman AM, Leenen EJ, Koopmans MP: Molecular detection of Norwalk-like caliciviruses in sewage. Appl Environ Microbiol 1999, 65: 5624-5627.PubMed CentralPubMedGoogle Scholar
- Pinto RM, Villena C, Le Guyader F, Guix S, Caballero S, Pommepuy M, Bosch A: Astrovirus detection in wastewater samples. Water Sci Technol 2001, 43: 73-76.PubMedGoogle Scholar
- Formiga-Cruz M, Hundesa A, Clemente-Casares P, Albinana-Gimenez N, Allard A, Girones R: Nested multiplex PCR assay for detection of human enteric viruses in shellfish and sewage. J Virol Methods 2005, 125: 111-118. 10.1016/j.jviromet.2005.01.009View ArticlePubMedGoogle Scholar
- Mustafa H, Palombo EA, Bishop RF: Improved sensitivity of astrovirus-specific RT-PCR following culture of stool samples in CaCo-2 cells. J Clin Virol 1998, 11: 103-107. 10.1016/S1386-6532(98)00049-XView ArticlePubMedGoogle Scholar
- Wang D, Urisman A, Liu YT, Springer M, Ksiazek TG, Erdman DD, Mardis ER, Hickenbotham M, Magrini V, Eldred J, Latreille JP, Wilson RK, Ganem D, DeRisi JL: Viral discovery and sequence recovery using DNA microarrays. PLoS Biol 2003, 1: E2. 10.1371/journal.pbio.0000002PubMed CentralView ArticlePubMedGoogle Scholar
- Swofford DL: PAUP*. Phylogenetic Anaylsis Using Parsimony (*and other methods). Sunderland, Massachusetts: Sinauer Associates; 1998.Google Scholar
- Calgua B, Mengewein A, Grunert A, Bofill-Mas S, Clemente-Casares P, Hundesa A, Wyn-Jones AP, Lopez-Pila JM, Girones R: Development and application of a one-step low cost procedure to concentrate viruses from seawater samples. J Virol Methods 2008, 153: 79-83. 10.1016/j.jviromet.2008.08.003View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.