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The three Aedes aegypti mosquito strains tested were susceptible to DENV-2 Jam1409 midgut infection and dissemination, as determined by detection of DENV-2 antigen in midguts and head tissues at day 14 post blood infection (dpi) (Figure 1). The midgut infection rates (MIR) were similar between Chetumal (87 ± 8%) and D2S3 (90 ± 7%), while they were lower in Rex-D (70 ± 7%) mosquitoes. The dissemination rates (DR) in Chetumal mosquitoes were greater (78 ± 10%) than in the laboratory-adapted Rex-D mosquitoes (57 ± 10%). As expected, the greatest dissemination rates were observed in the D2S3 mosquitoes (90 ± 7%). An ANOVA-one way analysis indicated that means were significantly different for MIR (P = 0.006) as well as for DR (P = 0.002) among the three mosquito populations. The MIR and DR between Chetumal and D2S3 mosquitoes did not differ significantly.
After ingestion of DENV-2, midguts were the first tissues to become infected. At 2 dpi, a few individual infected epithelial cells were distinguishable in ~30% of midguts. By day 3, midgut infection foci involved multiple cells. Infection spread laterally from this initial stage to the neighboring cells (probably due to virus release by budding as observed in invertebrate cells), frequently involving the entire organ by 7–10 dpi. Then, starting at day 10, but more noticeably at 14 dpi, the amount of viral antigen in the midgut began to decline. At 21 dpi viral antigen was almost undetectable, by either the polyclonal or monoclonal anti-dengue antibodies, in midguts of Chetumal (Figure 2) and in Rex-D control mosquitoes (data not shown). In contrast, other organs and tissues, including fat body, salivary glands, and nervous system in the same Chetumal mosquitoes contained large amounts of virus antigen during the time course.
DENV-2 + sense RNA as well as infectious virus in Chetumal mosquito midguts was quantified by Q-PCR and end point titration, respectively, at 7, 14 and 21 dpi. In this time frame, the viral RNA remained stable (between 7.2 to 7.6 Log10 RNA copy/midgut) as determined by Q-PCR (Figure 3-A). In contrast, between 7 and 21 dpi, virus titers decreased from 6 to 4 Log10 TCID50/midgut (P 3-B).
Analysis of mosquitoes between 2 and 10 dpi revealed the presence of DENV-2 antigen in portions of the tracheal system in 35 ± 5% of blood fed mosquitoes. The earliest detection of DENV-2 antigen in the tracheal system occurred at 2 dpi, continued at 3, 4, 5, and 7 dpi and then decreased notably. Viral antigen was not detected through out the tracheal system; rather it was confined to small sections of trachea mostly from the abdominal area and it displayed a characteristic distribution pattern (Figure 4). The presence of viral antigen in trachea and virus dissemination out of the midgut in Chetumal mosquitoes were strongly correlated at 2, 3 and 5 dpi (Table 1). This association was gone by 7 dpi (Table 1) and thereafter (data not shown). The criterion for disseminated infection was the presence of viral antigen in any organ or tissue other than the midgut or the tracheal system: early dissemination was detected most frequently in abdominal and throracic fat body, salivary glands and nerve cells and tissues. In contrast to the Chetumal mosquitoes, viral antigen was rarely associated with trachea of infected Rex-D mosquitoes.
DENV-2 was detected in the salivary glands of 36% of blood fed Chetumal mosquitoes at 4 dpi. In the Rex-D mosquitoes, this salivary gland infection rate did not occur until 10 dpi and at 7 dpi only 5).
The tropisms of DENV-2 Jam1409 to organ-tissues other than midguts and salivary glands were determined over time in Chetumal mosquitoes. DENV-2 antigen was detected in head tissues at 5 dpi, and antigen continued to accumulate in these tissues throughout the EIP. Viral antigen was detected in fat body of the abdominal, thoracic or cephalic regions consistently throughout the time course. DENV-2 dissemination to abdominal fat body was detected in 35 % of mosquitoes between 2 and 3 dpi (Figure 6, panel A) and in 62% of abdominal tissues at the other time points. Thus, fat body represents a major site for DENV-2 replication in the vector. Interestingly the fat body also plays a key role in triggering the insect immune response against pathogens. Hence, it would be important to determine potential effects of virus infection of the fat body on vector competence.
The musculature surrounding the midgut did not contain virus antigen at any of the examined time points in any of the mosquito populations included in our study (Figure 6-C). Other organs and infected tissues included: trachea, esophagus (Figure 6-E), hemocytes (Figure 6-F), ommatidia of the compound eye (Figure 6-G), nerve tissue (Figure 6-H), and malphigian tubules (Figure 6-I). Occasionally DENV-2 antigen was observed in anterior midgut (Figure 6-D) and not in the hindgut (Figure 6-I) or cardia (Figure 6-E). Taken together, these data revealed the general kinetics of infection and tropisms of DENV-2 in mosquito as shown in Figure 7.
To establish the overall kinetics of DENV-2 replication, viral titers were determined by plaque assay of individual mosquitoes at different time points (Figure 8). DENV-2 titers increased over time and ranged from 2.2 to 5.5 Log10 PFU/mosquito. Infectious virus was first detectable at 3 dpi. Two slight drops in the overall viral titers were observed: one at 14 dpi and the other at 21 dpi. Kinetics of virus replication during the DENV-2 infection at various time points are shown in Figure 8.
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