First Record of Leucocytozoon (Haemosporida: Leucocytozoidae) in Amazonia: Evidence for Rarity in Neotropical Lowlands or Lack of Sampling for This Parasite Genus?

Birds harbor an astonishing diversity of haemosporidian parasites belonging to the genera Haemoproteus, Leucocytozoon, and Plasmodium. Currently there are more than 250 morphologically described avian haemosporidian species and 2,828 unique lineages found in virtually all avian clades and zoogeographic regions, except for Antarctica. Our report is based on PCR and microscopic screening of 1,302 individual avian samples from Brazil to detect the underrepresented genus Leucocytozoon. This survey primarily focuses on passerine birds collected from Amazonia, the Atlantic Rain Forest, and Pantanal. We also summarize studies conducted in Brazil that report haemosporidian prevalence using both microscopy and molecular tools and present for the first time a record of Leucocytozoon infecting an avian host population in Amazonia. Based on our findings, we suggest that high average temperatures may be constraining both the distribution and diversity of Leucocytozoon in lowland tropical South America.

Sequence and secondary structure analysis of the 5'-terminal region of flavivirus genome RNA.

The 5'-terminal noncoding region sequences were determined for the genome RNAs of seven strains of St. Louis encephalitis virus (SLEV) and one strain of West Nile virus (WNV) using a single synthetic cDNA primer complementary to the 5'-terminus of the coding region of a strain of WNV RNA. The 5'-terminal sequences obtained for the SLEV and WNV RNAs were compared with published sequences for yellow fever virus (YFV), Murray Valley encephalitis virus (MVEV), and dengue virus. While only short regions within the 5'-noncoding sequence were conserved among different flavivirus RNAs, significant homology was observed in this region among members of the same flavivirus subgroup and almost complete conservation was observed between different strains of the same virus. For example, seven strains of SLE, isolated from different geographic locations over a 17-year period and differing in their neurovirulence phenotype, contained only two to four nucleotide changes in the 5'-noncoding region. Interestingly, each of three low-virulence strains shared the same unique base substitution at position 16. Secondary structures predicted to be formed by the 5'-termini of each of the different flavivirus genome RNAs were of similar size and shape, in each case consisting of a stem with a small top loop and a larger side loop. The prediction of a common structure among a number of different flaviviruses, despite the lack of extensive sequence homology, suggests that this secondary structure is functionally important. An additional stem and loop structure is predicted to be formed in the region spanning the translation initiation codon. This structure showed significantly less conservation of size and shape than the 5'-terminal secondary structure.

The 3'-nucleotides of flavivirus genomic RNA form a conserved secondary structure.

The terminal noncoding regions of viral RNA genomes are presumed to contain signal sequences and sometimes also secondary structures involved in regulating viral RNA synthesis. Such signals would be expected to be highly conserved among related viruses. In order to identify replication signal features for flaviviruses we have compared the 3'-terminal nucleotide sequences of West Nile virus (WNV), Saint Louis encephalitis (SLE) virus, and yellow fever virus (YFV) genome RNAs. The existence of a stable 3'-terminal secondary structure was previously predicted by a cDNA sequence obtained from YFV genome RNA. We have confirmed the existence of this structure by direct RNA sequencing methods. Even though the size and shape of the 3'-terminal secondary structure is highly conserved, sequence conservation is restricted to the loop regions of the secondary structure and to 27 nucleotides immediately adjacent to the 5' side of the structure. The regions of conserved sequence represent likely signals for viral polymerase recognition and binding. However, the preservation of the configuration of the secondary structure by a means other than sequence conservation indicate that this structure is important for the survival of the virus. A WNV mutant, which replicates progeny genome RNA more efficiently than parental WNV, was found to have a 3'-genomic sequence identical to that of its parent virus. The sequence change conferring the phenotype of this mutant is therefore located in another region of the genome.