Analysis of codon usage bias in potato virus Y non-recombinant strains.

Potato virus Y (PVY) is a member of the genus Potyvirus, family Potyviridae, is considered one of the most devastating pest affecting economically important crops, such as potato, tobacco, tomato and pepper, representing a serious threat due to high incidence and worldwide distribution. Its economic significance as well as it biological and molecular complexities have aroused great attention, thus several studies have explore it genetic characteristics. However, little is known about PVY codon usage. To shed light on the relation of codon usage among viruses and their hosts is extremely important to understand virus survival, fitness and evolution. In this study, we performed a comprehensive analysis of codon usage and composition of PVY non-recombinant strains (PVYN-NA, PVYEu-N, PVYO, PVYO5, PVYC) based on 130 complete open reading frame sequences extracted from public databases. Furthermore, similarities between the synonymous codon usage of PVY and its main hosts were investigated. The results obtained in the current study suggest that the overall codon usage among PVY genotypes is similar and slightly biased. PVY codon usage is strongly influenced by mutational bias, but also by G + C compositional constraint and dinucleotide composition. Furthermore, similarities among codon usage preferences between PVY strains and analyzed hosts were observed.

The clinically approved antiviral drug sofosbuvir inhibits Zika virus replication.

Zika virus (ZIKV) is a member of the Flaviviridae family, along with other agents of clinical significance such as dengue (DENV) and hepatitis C (HCV) viruses. Since ZIKV causes neurological disorders during fetal development and in adulthood, antiviral drugs are necessary. Sofosbuvir is clinically approved for use against HCV and targets the protein that is most conserved among the members of the Flaviviridae family, the viral RNA polymerase. Indeed, we found that sofosbuvir inhibits ZIKV RNA polymerase, targeting conserved amino acid residues. Sofosbuvir inhibited ZIKV replication in different cellular systems, such as hepatoma (Huh-7) cells, neuroblastoma (SH-Sy5y) cells, neural stem cells (NSC) and brain organoids. In addition to the direct inhibition of the viral RNA polymerase, we observed that sofosbuvir also induced an increase in A-to-G mutations in the viral genome. Together, our data highlight a potential secondary use of sofosbuvir, an anti-HCV drug, against ZIKV.

Aichi Virus Positivity in HIV-1 Seropositive Children Hospitalized with Diarrheal Disease.

Aichi viruses (AiV) have been detected in patients with diarrheal diseases (DD). The aim of this study was to assess AiV infection rates in hospitalized children with DD, including 123 HIV-1 seropositive and 125 HIV-1 seronegative patients, in two public pediatric hospitals in Rio de Janeiro, Brazil. AiV was investigated by nested RT-PCR. The AiV-positive samples were also tested for specie A rotavirus, norovirus, astrovirus, enteric adenovirus and bocavirus in order to assess co-infections. AiV parcial genome sequencing and phylogenetic analyses were performed. AiV were detected in 9/123 (7.32%) of the HIV-1 seropositive subjects and 1/125 (0.8%) of the HIV seronegative patients with DD (p = 0.019). The phylogenetic analysis of positive samples disclosed that: i) 13 samples were characterized as genotype A, with one of them being from the HIV-1 seronegative patient; ii) one sample from a HIV-1 seropositive patient was characterized as genotype B. AiV genotype A was grouped into 3 genetic clusters. Data suggest that AiV may be an opportunistic pathogen infecting children with AIDS and DD.

Phylogenetic analysis of G1P[6] group A rotavirus strains detected in Northeast Brazilian children fully vaccinated with Rotarix™.

In 2009 the World Health Organization recommended the use of group A rotavirus (RVA) vaccines in all national immunization programs (NIPs) in order to control severe RVA gastroenteritis disease. In Brazil, Rotarix™ was introduced in the NIP in March 2006, and a significant reduction in mortality rates among children ≤ 5 years old was observed, especially in the Northern and Northeastern Brazil. In the current study the 11 gene segments of six Brazilian G1P[6] RVA strains, isolated in 2009 and 2010 from vaccinated children, were analyzed in order to investigate if the genetic composition of these strains might help to elucidate why they were able to cause acute gastroenteritis in vaccinated children. All six Brazilian RVA strains revealed a complete Wa-like genotype constellation: G1-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1. Phylogenetic analysis showed that all six strains were nearly identical and showed a close genetic relationship with contemporary typical human Wa-like RVA strains. These results suggests that the fact that these strains were able to cause acute gastroenteritis in vaccinated children is likely not due to the genetic background of the strains, but rather to other factors such as host relating factors, co-infecting pathogens or vaccine efficacy. P[6] RVA strains are detected rather occasionally in humans in most regions of the world, except for South Asia and Sub-Saharan Africa. However, recently two studies conducted in Brazil showed the circulation of G12P[6] and G2P[6]. This is the first report on the detection and complete genome analyses of G1P[6] RVA strains in Brazil. Surveillance studies will be crucial to further investigate the prevalence of this genotype in the Brazilian population, and the efficacy of current licensed vaccines, which do not contain the P[6] genotype.

Use of acridine orange staining for the detection of rotavirus RNA in polyacrylamide gels.

Acridine orange is a metachromatic intercalator used extensively in histochemistry to differentiate double- from single-stranded (ds, ss) nucleic acid by the emission of green and red fluorescence, respectively, under ultraviolet light. In the present study we standardised a protocol in order to use acridine orange to detect rotavirus ds RNA in polyacrylamide gels and compared it to silver and ethidium bromide staining. We demonstrated that the simplest and best condition was attained when gels containing rotavirus ds RNA bands, stained in green, were treated with 4.3 microM acridine orange after electrophoresis and destained with distilled water pH 6 at 37 degrees C. Under this protocol, rotavirus RNA concentration was calculated and the mean minimum amounts of nucleic acid detected by acridine orange, ethidium bromide, and silver staining were 26.0 +/- 4.29, 15.6 +/- 1.48 and 1.06 +/- 0.11 ng, respectively. The comparison of acridine orange sensitivity with ethidium bromide and silver staining, for 25 field strains of rotavirus and one cell-adapted strain (SA11), demonstrated concurrent results in 80% of the specimens. Red colour emission resulting from the interaction of acridine orange with ss nucleic acid was also shown by testing denatured 0.5 kb HindIII digest of lambda phage DNA. Furthermore, it was demonstrated that rotavirus ds RNA could be used for reverse transcription activity, followed by PCR amplification, after acridine orange staining. In conclusion, although acridine orange is less sensitive than ethidium bromide and silver staining, its practicality, low cost, metachromatic properties, and its non-interference on RT-PCR should be considered. It is suggested the use of acridine orange as an appropriate stain for various purposes in virology, as well as for the molecular biology of nucleic acid.