Dengue Virus Serotype 4, Roraima State, Brazil

bred outdoors in malaria-endemic areas is not uncommon. However, with the exception of P. knowlesi, it is my understanding that malarial organisms found in cynomolgus monkeys do not pose a major zoonotic concern (although this can always change). Furthermore, it is my understanding that P. vivax does not infect macaques, including cynomolgus monkeys. Other malarial parasites of cynomolgus monkeys, apart from P. knowlesi, may include P. cynomologi, P. inui, P. fi eldi, and P. coatneyi. A recent publication reported that in wild-source cynomolgus monkeys in Malaysia, >90% of the animals tested were positive for >1 Plasmodium species. Furthermore, >80% of samples from these animals were positive by specifi c PCR for >1 of these organisms (2). Using PCR for Plasmodium spp. identifi cation, I have tested newly imported research cynomolgus monkeys from various breeding centers in China. I can confi rm that some animals have subclinical malarial infections. Except for the report by Li et al. (1), I am unaware of other reports of P. vivax in cynomolgus monkeys. It would be interesting to confi rm the presence of this organism by using PCR primers specifi c for Plasmodium spp. My questions to the authors relate to the test method used in their study. Was an ELISA for detecting P. vivax antibodies the only diagnostic method used to identify this parasite? It may be useful to re-address the specifi city of this test in differentiating various Plasmodium spp. Until these issues are clearly addressed, their reported results are not reliable.

Temporão et al. conducted phylogenetic analysis of envelope gene sequences and concluded that 3 samples of DENV from Roraima in 2010 were DENV-4, genotype I (1). Unfortunately, the authors mistakenly labeled Asian strains (Thailand-1978 and-1985) as genotype II, and American strains (e.g., Brazil-1982) as genotype I. Those DENV-4 strains isolated in Roraima in 2010 in fact belong to genotype II (2,3). We had previously analyzed 2 samples isolated from Roraima in 2010 by using C/prM nucleotide sequencing and maximumlikelihood phylogenetic reconstruction. Our results, presented at the XXI National Meeting of Brazilian Society for Virology in October 2010, show that both isolates are indeed genotype II (3). Nucleotide sequences are available in GenBank under accession nos. HQ822125 and HQ822126.
Temporão et al. also concluded that because only genotype II (reported as genotype I) was identifi ed in their samples, "[it] excludes the possibility that Asian genotypes previously circulated in Brazil." Beyond its obviously fl awed logic, we believe that this statement lacks scientifi c support; DENV-4 genotype I, closely related to Chinese and Philippine strains, has in fact been shown to occur in the city of Manaus, ≈800 km south of Boa Vista, as reported in 2 recent articles (4,5). Circulation of DENV-4 genotype I in northern Brazil, probably related to increasingly intense trade with Asian countries, may be sporadic and geographically limited as yet (5) by laboratory contamination with Asian genotype I. Possible introduction and detection of Asian DENV-4 strains in Brazil should not be ignored because the possibility of multiple introduction events in the country resulting from intense transit of people and commercial activities across Brazil from the Caribbean and Asian regions poses a real risk. However, at this time, only genotype II has been isolated and genetically characterized (1). The previously published articles lack strong and reliable scientifi c evidence.

Novel Hepatitis E Virus Genotype in Norway Rats, Germany
To the Editor: We read with interest the article by Johne et al. about 2 novel hepatitis E virus (HEV) isolates in Norway rats in Germany (1). Some points in the report deserve comment.
First, because of degeneracy of the genetic code, HEV amino acid sequences are more conserved than nucleotide sequences. For instance, although the open reading frame 2 of the avian HEV isolate (GenBank accession no. AY535004) has only 65% nt sequence homology to that of the swine HEV isolate swGX32 (GenBank accession no. EU366959), their amino acid sequences shared >90% identity. However, the table in (1) indicated the amino acid sequence homologies between the novel and previous HEV isolates were similar to (some even lower than) the nucleotide sequence homologies. These low sequence identities of the capsid proteins between the novel and previous HEVs may explain why no HEV antibody-positive rat was found in the initial serologic screening with a commercial genotype 1-based ELISA. Furthermore, we wonder how the novel antigen in the hepatocytes could react with the anti-HEV serum in the immunohistochemical staining.
Second, the authors stated they determined the entire virus genome by using a previously described method (2). The primers in that method were designed to amplify a genotype 3 HEV isolate with low (55.7%) sequence homology to the 2 novel HEV isolates and therefore cannot amplify their sequences. We ask the authors to list the new primer sequences they used, which will help determine the full viral genome if this virus is found in other regions or animal species.
Suggesting the rabbit HEV sequences may be representative genotype 3 isolates is not yet appropriate because not enough research has yet determined whether rabbit HEV infects other species. Therefore, the rabbit HEV sequence FJ906895 should not be listed as representative genotype 3 isolate as in Figure 1 in (1). Also, the swine isolate DQ450072 should not be listed as a representative genotype 4 isolate; a recent report indicated it was a recombinant produced between genotypes 3 and 4 isolates (3).