Computational protocol: Ocozocoautla de Espinosa Virus and Hemorrhagic Fever, Mexico

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Protocol publication

[…] Kidney samples from the 3 antibody-positive Mexican deer mice, Mexican deer mouse TK93314 () and 8 other antibody-negative Mexican deer mice, 18 southern pygmy mice (Baiomys musculus), and 11 Jaliscan cotton rats (Sigmodon mascotensis) were tested for arenavirus by cultivation in monolayers of Vero E6 cells (). The 3 antibody-positive deer mice (TK93319, TK93321, TK93325) and 38 antibody-negative rodents were captured on July 16, 2000, at a locality (Universal Transverse Mercator coordinates 15–451772E, 1864243N; elevation 1,021 m) in the municipality of Ocozocoautla de Espinosa ().Kidney samples from the 3 antibody-positive animals also were tested for arenavirus nucleocapsid (N) protein gene RNA. First-strand cDNA was synthesized by using SuperScript III Reverse Transcriptase (Invitrogen Corp., Carlsbad, CA, USA) and oligont 19C-cons (). The first-round PCR used MasterTaq Kit (5 PRIME, Inc., Gaithersburg, MD, USA) and 19C-cons and either AVNP42 (5′-GCCGCGGACTGGGAGGGCA-3′) or AVNP122 (5′-GCCGCGGACTGGGGAGGCACTG-3′). The second-round (heminested) PCR used MasterTaq Kit and either AVNP42 and oligont 1010C (), AVNP122 and 1010C, or AVNP115 (5′-CCAATATAAGGCCAACCATCG-3′) and AVNP149 (5′-CGCACAGTGGATCCTAGGCATAGTGTC-3′). The nucleotide sequence of a 3,380-nt fragment of the small genomic segment of arenavirus AV B1030026 (GenBank accession no. JN897398) was then determined from the first-strand cDNA from antibody-positive deer mouse TK93325 by using a series of 3 heminested PCRs. The 3,380-nt fragment extended from within the 5′ noncoding region, through the glycoprotein precursor (GPC) gene, intergenic region, and N protein gene, and into the 3′ noncoding region.Analyses of GPC sequences, N protein sequences, and nucleotide sequences included AV B1030026, 8 other viruses from North America, and 15 viruses from South America (). Sequences in each amino acid sequence dataset were aligned by using ClustalW version 2.0.12 (). The sequences in each nucleotide sequence dataset were aligned manually, and alignment was guided by the corresponding computer-generated amino acid sequence alignment. Sequence nonidentities were equivalent to uncorrected (p) distances.Phylogenetic analyses of nucleotide sequences were conducted by using MRBAYES version 3.1.2 () and programs in PAUP* (). Bayesian analyses used the general time reversible + proportion invariant + Γ model with a site-specific gamma distribution and the following options in MRBAYES version 3.1.2: two simultaneous runs of 4 Markov chains, 10 million generations, and sample frequency every 1,000th generation. The first 1,000 trees were discarded after review of the likelihood scores, convergence statistics, and potential scale reduction factors. A consensus tree (50% majority rule) was constructed from the remaining trees. Probability values in support of the clades were calculated a posteriori, and clades with probability values >0.95 were considered supported by the data (). […]

Pipeline specifications

Software tools Clustal W, MrBayes, PAUP*
Application Phylogenetics
Organisms Mus musculus, Homo sapiens
Diseases Hemorrhagic Fever, American, HIV Infections