Pulmonary lesions induced by SARS-CoV-2 infection in domestic cats.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019, which ranges from fatal disease in some to mild or subclinical in most affected individuals. Many recovered human patients report persistent respiratory signs; however, lung disease in post-acute infection is poorly understood. Our objective was to describe histologic lung lesions and viral loads following experimental SARS-CoV-2 infection in 11 cats. Microscopic evaluation at 3, 6, 10, or 28 days postinoculation (DPI) identified mild to moderate patchy interstitial pneumonia, bronchiolar epithelial damage, and occlusive histiocytic bronchiolitis. Based on immunohistochemistry, alveolar septal thickening was due to CD204-positive macrophages, fewer B and T lymphocytes, type II pneumocytes, and capillary proliferation with a relative dearth of fibrosis. In blood vessel endothelium, there was reactive hypertrophy or vacuolar degeneration and increased MHC II expression at all time points. Unexpectedly, one cat from the 28 DPI group had severe subacute regionally extensive lymphohistiocytic pneumonia with multifocal consolidation, vasculitis, and alveolar fibrin. Reverse transcriptase-quantitative polymerase chain reaction identified SARS-CoV-2 RNA within the lung at 3 and 6 DPI, and viral RNA was below the limit of detection at 10 and 28 DPI, suggesting that pulmonary lesions persist beyond detection of viral RNA. These findings clarify our comparative understanding of disease induced by SARS-CoV-2 and suggest that cats can serve as an informative model to study post-acute pulmonary sequelae.

Parameters of Mosquito-Enhanced West Nile Virus Infection.

UNLABELLED: The arthropod-borne West Nile virus (WNV) emerged in New York State in 1999 and quickly spread throughout the United States. Transmission is maintained in an enzootic cycle in which infected mosquitoes transmit the virus to susceptible hosts during probing and feeding. Arthropod-derived components within the viral inoculum are increasingly acknowledged to play a role in infection of vertebrate hosts. We previously showed that Culex tarsalis mosquito saliva and salivary gland extract (SGE) enhance the in vivo replication of WNV. Here, we characterized the effective dose, timing, and proximity of saliva and SGE administration necessary for enhancement of WNV viremia using a mouse model. Mosquito saliva and SGE enhanced viremia in a dose-dependent manner, and a single mosquito bite or as little as 0.01 µg of SGE was effective at enhancing viremia, suggesting a potent active salivary factor. Viremia was enhanced when SGE was injected in the same location as virus inoculation from 24 h before virus inoculation through 12 h after virus inoculation. These results were confirmed with mosquito saliva deposited by uninfected mosquitoes. When salivary treatment and virus inoculation were spatially separated, viremia was not enhanced. In summary, the effects of mosquito saliva and SGE were potent, long lasting, and localized, and these studies have implications for virus transmission in nature, where vertebrate hosts are fed upon by both infected and uninfected mosquitoes over time. Furthermore, our model provides a robust system to identify the salivary factor(s) responsible for enhancement of WNV replication. IMPORTANCE: Mosquito-borne viruses are a significant class of agents causing emerging infectious diseases. WNV has caused over 18,000 cases of neuroinvasive disease in the United States since its emergence. We have shown that Culex tarsalis mosquito saliva and SGE enhance the replication of WNV. We now demonstrate that saliva and SGE have potent, long-lasting, and localized effects. Our model provides a robust system to identify the salivary factor(s) and characterize the mechanism responsible for enhancement of WNV replication. These studies could lead to the identification of novel prophylactic or treatment options useful in limiting the spread of WNV, other mosquito-borne viruses, and the diseases that they cause.

West Nile virus methyltransferase domain interacts with protein kinase G.

BACKGROUND: The flaviviral nonstructural protein 5 (NS5) is a phosphoprotein, though the precise identities and roles of many specific phosphorylations remain unknown. Protein kinase G (PKG), a cGMP-dependent protein kinase, has previously been shown to phosphorylate dengue virus NS5. METHODS: We used mass spectrometry to specifically identify NS5 phosphosites. Co-immunoprecipitation assays were used to study protein-protein interactions. Effects on viral replication were measured via replicon system and plaque assay titering. RESULTS: We identified multiple sites in West Nile virus (WNV) NS5 that are phosphorylated during a WNV infection, and showed that the N-terminal methyltransferase domain of WNV NS5 can be specifically phosphorylated by PKG in vitro. Expressing PKG in cell culture led to an enhancement of WNV viral production. We hypothesized this effect on replication could be caused by factors beyond the specific phosphorylations of NS5. Here we show for the first time that PKG is also able to stably interact with a viral substrate, WNV NS5, in cell culture and in vitro. While the mosquito-borne WNV NS5 interacted with PKG, tick-borne Langat virus NS5 did not. The methyltransferase domain of NS5 is able to mediate the interaction between NS5 and PKG, and mutating positive residues in the αE region of the methyltransferase interrupts the interaction. These same mutations completely inhibited WNV replication. CONCLUSIONS: PKG is not required for WNV replication, but does make a stable interaction with NS5. While the consequence of the NS5:PKG interaction when it occurs is unclear, mutational data demonstrates that this interaction occurs in a region of NS5 that is otherwise necessary for replication. Overall, the results identify an interaction between virus and a cellular kinase and suggest a role for a host kinase in enhancing flaviviral replication.

Keratinocytes are cell targets of West Nile virus in vivo.

West Nile virus (WNV) replicates in the skin; however, cell targets in the skin have not been identified. In the current studies, WNV infected the epidermis and adnexal glands of mouse skin, and the epidermal cells were identified as keratinocytes by double labeling for WNV antigen and keratin 10. Inoculation of mice with WNV replicon particles resulted in high levels of replication in the skin, suggesting that keratinocytes are an initial target of WNV. In addition, primary keratinocytes produced infectious virus in vitro. In conclusion, keratinocytes are cell targets of WNV in vivo and may play an important role in pathogenesis.

Mosquito saliva causes enhancement of West Nile virus infection in mice.

West Nile virus (WNV) is transmitted to vertebrate hosts primarily by infected Culex mosquitoes. Transmission of arboviruses by the bite of infected mosquitoes can potentiate infection in hosts compared to viral infection by needle inoculation. Here we examined the effect of mosquito transmission on WNV infection and systematically investigated multiple factors that differ between mosquito infection and needle inoculation of WNV. We found that mice infected with WNV through the bite of a single infected Culex tarsalis mosquito exhibited 5- to 10-fold-higher viremia and tissue titers at 24 and 48 h postinoculation and faster neuroinvasion than mice given a median mosquito-inoculated dose of WNV (10(5) PFU) by needle. Mosquito-induced enhancement was not due to differences in inoculation location, because additional intravenous inoculation of WNV did not enhance viremia or tissue titers. Inoculation of WNV into a location where uninfected mosquitoes had fed resulted in enhanced viremia and tissue titers in mice similar to those in mice infected by a single infected mosquito bite, suggesting that differences in where virus is deposited in the skin and in the virus particle itself were not responsible for the enhanced early infection in mosquito-infected mice. In addition, inoculation of mice with WNV mixed with salivary gland extract (SGE) led to higher viremia, demonstrating that mosquito saliva is the major cause of mosquito-induced enhancement. Enhanced viremia was not observed when SGE was inoculated at a distal site, suggesting that SGE enhances WNV replication by exerting a local effect. Furthermore, enhancement of WNV infection still occurred in mice with antibodies against mosquito saliva. In conclusion, saliva from C. tarsalis is responsible for enhancement of early WNV infection in vertebrate hosts.

Nonconsensus West Nile virus genomes arising during mosquito infection suppress pathogenesis and modulate virus fitness in vivo.

West Nile virus (WNV) is similar to other RNA viruses in that it forms genetically complex populations within hosts. The virus is maintained in nature in mosquitoes and birds, with each host type exerting distinct influences on virus populations. We previously observed that prolonged replication in mosquitoes led to increases in WNV genetic diversity and diminished pathogenesis in mice without remarkable changes to the consensus genome sequence. We therefore sought to evaluate the relationships between individual and group phenotypes in WNV and to discover novel viral determinants of pathogenesis in mice and fitness in mosquitoes and birds. Individual plaque size variants were isolated from a genetically complex population, and mutations conferring a small-plaque and mouse-attenuated phenotype were localized to the RNA helicase domain of the NS3 protein by reverse genetics. The mutation, an Asp deletion, did not alter type I interferon production in the host but rendered mutant viruses more susceptible to interferon compared to wild type (WT) WNV. Finally, we used an in vivo fitness assay in Culex quinquefasciatus mosquitoes and chickens to determine whether the mutation in NS3 influenced fitness. The fitness of the NS3 mutant was dramatically lower in chickens and moderately lower in mosquitoes, indicating that RNA helicase is a major fitness determinant of WNV and that the effect on fitness is host specific. Overall, this work highlights the complex relationships that exist between individual and group phenotypes in RNA viruses and identifies RNA helicase as an attenuation and fitness determinant in WNV.

An adenosine nucleoside inhibitor of dengue virus.

Dengue virus (DENV), a mosquito-borne flavivirus, is a major public health threat. The virus poses risk to 2.5 billion people worldwide and causes 50 to 100 million human infections each year. Neither a vaccine nor an antiviral therapy is currently available for prevention and treatment of DENV infection. Here, we report a previously undescribed adenosine analog, NITD008, that potently inhibits DENV both in vitro and in vivo. In addition to the 4 serotypes of DENV, NITD008 inhibits other flaviviruses, including West Nile virus, yellow fever virus, and Powassan virus. The compound also suppresses hepatitis C virus, but it does not inhibit nonflaviviruses, such as Western equine encephalitis virus and vesicular stomatitis virus. A triphosphate form of NITD008 directly inhibits the RNA-dependent RNA polymerase activity of DENV, indicating that the compound functions as a chain terminator during viral RNA synthesis. NITD008 has good in vivo pharmacokinetic properties and is biologically available through oral administration. Treatment of DENV-infected mice with NITD008 suppressed peak viremia, reduced cytokine elevation, and completely prevented the infected mice from death. No observed adverse effect level (NOAEL) was achieved when rats were orally dosed with NITD008 at 50 mg/kg daily for 1 week. However, NOAEL could not be accomplished when rats and dogs were dosed daily for 2 weeks. Nevertheless, our results have proved the concept that a nucleoside inhibitor could be developed for potential treatment of flavivirus infections.

Persistence of virus-specific immune responses in the central nervous system of mice after West Nile virus infection.

BACKGROUND: West Nile virus (WNV) persists in humans and several animal models. We previously demonstrated that WNV persists in the central nervous system (CNS) of mice for up to 6 months post-inoculation. We hypothesized that the CNS immune response is ineffective in clearing the virus. RESULTS: Immunocompetent, adult mice were inoculated subcutaneously with WNV, and the CNS immune response was examined at 1, 2, 4, 8, 12 and 16 weeks post-inoculation (wpi). Characterization of lymphocyte phenotypes in the CNS revealed elevation of CD19+ B cells for 4 wpi, CD138 plasma cells at 12 wpi, and CD4+ and CD8+ T cells for at least 12 wpi. T cells recruited to the brain were activated, and regulatory T cells (Tregs) were present for at least 12 wpi. WNV-specific antibody secreting cells were detected in the brain from 2 to 16 wpi, and virus-specific CD8+ T cells directed against an immunodominant WNV epitope were detected in the brain from 1 to 16 wpi. Furthermore, these WNV-specific immune responses occurred in mice with and without acute clinical disease. CONCLUSIONS: Virus-specific immune cells persist in the CNS of mice after WNV infection for up to 16 wpi.

Exclusion of West Nile virus superinfection through RNA replication.

Superinfection exclusion is the ability of an established viral infection to interfere with a second viral infection. Using West Nile virus (WNV) as a model, we show that replicating replicons in BHK-21 cells suppress subsequent WNV infection. The WNV replicon also suppresses superinfections of other flaviviruses but not nonflaviviruses. Mode-of-action analysis indicates that the exclusion of WNV superinfection occurs at the step of RNA synthesis. The continuous culturing of WNV in the replicon-containing cells generated variants that could overcome the superinfection exclusion. The sequencing of the selected viruses revealed mutations in structural (prM S90R or envelope E138K) and nonstructural genes (NS4a K124R and peptide 2K V9M). Mutagenesis analysis showed that the mutations in structural genes nonselectively enhance viral infection in both naïve and replicon-containing BHK-21 cells; in contrast, the mutations in nonstructural genes more selectively enhance viral replication in the replicon-containing cells than in the naïve cells. Mechanistic analysis showed that the envelope mutation functions through the enhancement of virion attachment to BHK-21 cells, whereas the 2K mutation (and, to a lesser extent, the NS4a mutation) functions through the enhancement of viral RNA synthesis. Furthermore, we show that WNV superinfection exclusion is reversible by the treatment of the replicon cells with a flavivirus inhibitor. The preestablished replication of the replicon could be suppressed by infecting the cells with the 2K mutant WNV but not with the wild-type virus. These results suggest that WNV superinfection exclusion is a result of competition for intracellular host factors that are required for viral RNA synthesis.

Mosquitoes inoculate high doses of West Nile virus as they probe and feed on live hosts.

West Nile virus (WNV) is transmitted to vertebrate hosts by mosquitoes as they take a blood meal. The amount of WNV inoculated by mosquitoes as they feed on a live host is not known. Previous estimates of the amount of WNV inoculated by mosquitoes (10(1.2)-10(4.3) PFU) were based on in vitro assays that do not allow mosquitoes to probe or feed naturally. Here, we developed an in vivo assay to determine the amount of WNV inoculated by mosquitoes as they probe and feed on peripheral tissues of a mouse or chick. Using our assay, we recovered approximately one-third of a known amount of virus inoculated into mouse tissues. Accounting for unrecovered virus, mean and median doses of WNV inoculated by four mosquito species were 10(4.3) PFU and 10(5.0) PFU for Culex tarsalis, 10(5.9) PFU and 10(6.1) PFU for Cx. pipiens, 10(4.7) PFU and 10(4.7) PFU for Aedes japonicus, and 10(3.6) PFU and 10(3.4) PFU for Ae. triseriatus. In a direct comparison, in vivo estimates of the viral dose inoculated by Cx. tarsalis were approximately 600 times greater than estimates obtained by an in vitro capillary tube transmission assay. Virus did not disperse rapidly, as >99% of the virus was recovered from the section fed or probed upon by the mosquito. Furthermore, 76% (22/29) of mosquitoes inoculated a small amount of virus ( approximately 10(2) PFU) directly into the blood while feeding. Direct introduction of virus into the blood may alter viral tropism, lead to earlier development of viremia, and cause low rates of infection in co-feeding mosquitoes. Our data demonstrate that mosquitoes inoculate high doses of WNV extravascularly and low doses intravascularly while probing and feeding on a live host. Accurate estimates of the viral dose inoculated by mosquitoes are critical in order to administer appropriate inoculation doses to animals in vaccine, host competence, and pathogenesis studies.

Molecular epidemiology of eastern equine encephalitis virus, New York.

Perpetuation, overwintering, and extinction of eastern equine encephalitis virus (EEEV) in northern foci are poorly understood. We therefore sought to describe the molecular epidemiology of EEEV in New York State during current and past epizootics. To determine whether EEEV overwinters, is periodically reintroduced, or both, we sequenced the E2 and partial NSP3 coding regions of 42 EEEV isolates from New York State and the Eastern Seaboard of the United States. Our phylogenetic analyses indicated that derived subclades tended to contain southern strains that had been isolated before genetically similar northern strains, suggesting southern to northern migration of EEEV along the Eastern Seaboard. Strong clustering among strains isolated during epizootics in New York from 2003-2005, as well as from 1974-1975, demonstrates that EEEV has overwintered in this focus. This study provides molecular evidence for the introduction of southern EEEV strains to New York, followed by local amplification, perpetuation, and overwintering.

A duplex real-time reverse transcriptase polymerase chain reaction assay for the detection of St. Louis encephalitis and eastern equine encephalitis viruses.

A duplex TaqMan real-time reverse transcriptase polymerase chain reaction (PCR) assay was developed for the detection of St. Louis encephalitis virus (SLEV) and eastern equine encephalitis virus (EEEV), for use in human and vector surveillance. The respective targets selected for the assay were the conserved NS5 and E1 genes of the 2 viruses. Because of the insufficient number of NS5 sequences from SLEV strains in the GenBank database, we determined the sequence of an approximately 1-kb region for each of 25 strains of SLEV to select primers and probes in a conserved region. Our assay has a sensitivity of 5 gene copies (gc)/reaction for EEEV and 10 gc/reaction for SLEV, and its performance is linear for at least 6 log(10) gc. The assay is specific and detected all strains of SLEV (69) and EEEV (12) that were tested. An internal control ensures detection of efficient nucleic acid extraction and possible PCR inhibition.

Bluetongue virus serotype 17 sequence variation associated with neutralization.

Bluetongue virus (BTV) is an insect-transmitted orbivirus of importance to the cattle and sheep industry. The VP2 protein, encoded by L2, contains neutralizing epitopes. Previously, a panel of neutralizing monoclonal antibodies (MAbs) to the BTV serotype 17 (BTV-17) prototype strain was generated and it was determined that the neutralization domain consists of three overlapping epitopes. Over 30 amino acid changes were found between a neutralized BTV-17 prototype strain and a non-neutralized BTV-17 198 strain. In this study, the L2 genes from eight additional strains, representing both the neutralized and non-neutralized groups of BTV-17, were sequenced to determine the degree of conservation of the previously characterized differences. Comparison of the deduced amino acid sequences showed that 91% (30/33) of the previously noted changes were conserved within each group. The sequence of the M5 gene that encodes VP5 was also examined, since this surface protein has also been shown to affect neutralization. No consistent changes were noted between the neutralized and non-neutralized groups of BTV-17 by analysis of the VP5 protein. Finally, the L2 sequences of five MAb neutralization escape mutants were determined to identify specific amino acids involved in neutralization and perhaps virulence. All five mutants contained 1-3 amino acid changes that were in close proximity to a previously described variable region. These amino acid changes likely define critical sites in the overlapping neutralization domains previously described. This is the first description of two BT virus populations that have distinct neutralization characteristics co-circulating in a defined geographical region.

A deep insight into the male and female sialotranscriptome of adult Culex tarsalis mosquitoes.

Previously, a Sanger-based sialotranscriptome analysis of adult female Culex tarsalis was published based on ∼2000 ESTs. During the elapsed 7.5 years, pyrosequencing has been discontinued and Illumina sequences have increased considerable in size and decreased in price. We here report an Illumina-based sialotranscriptome that allowed finding the missing apyrase from the salivary transcriptome of C. tarsalis, to determine several full-length members of the 34-62 kDa family, when a single EST has been found previously, in addition to identifying many salivary families with lower expression levels that were not detected previously. The use of multiple libraries including salivary glands and carcasses from male and female organisms allowed for an unprecedented insight into the tissue specificity of transcripts, and in this particular case permitting identification of transcripts putatively associated with blood feeding, when exclusive of female salivary glands, or associated with sugar feeding, when transcripts are found upregulated in both male and female glands.

Growth and adaptation of Zika virus in mammalian and mosquito cells.

The recent emergence of Zika virus (ZIKV) in the Americas coincident with increased caseloads of microcephalic infants and Guillain-Barre syndrome has prompted a flurry of research on ZIKV. Much of the research is difficult to compare or repeat because individual laboratories use different virus isolates, growth conditions, and quantitative assays. Here we obtained three readily available contemporary ZIKV isolates and the prototype Ugandan isolate. We generated stocks of each on Vero mammalian cells (ZIKVmam) and C6/36 mosquito cells (ZIKVmos), determined titers by different assays side-by-side, compared growth characteristics using one-step and multi-step growth curves on Vero and C6/36 cells, and examined plaque phenotype. ZIKV titers consistently peaked earlier on Vero cells than on C6/36 cells. Contemporary ZIKV isolates reached peak titer most quickly in a multi-step growth curve when the amplifying cell line was the same as the titering cell line (e.g., ZIKVmam titered on Vero cells). Growth of ZIKVmam on mosquito cells was particularly delayed. These data suggest that the ability to infect and/or replicate in insect cells is limited after growth in mammalian cells. In addition, ZIKVmos typically had smaller, more homogenous plaques than ZIKVmam in a standard plaque assay. We hypothesized that the plaque size difference represented early adaptation to growth in mammalian cells. We plaque purified representative-sized plaques from ZIKVmos and ZIKVmam. ZIKVmos isolates maintained the initial phenotype while plaques from ZIKVmam isolates became larger with passaging. Our results underscore the importance of the cells used to produce viral stocks and the potential for adaptation with minimal cell passages. In addition, these studies provide a foundation to compare current and emerging ZIKV isolates in vitro and in vivo.

Genomic, Recombinational and Phylogenetic Characterization of Global Feline Herpesvirus 1 Isolates.

Feline herpes virus type 1 (FHV-1) is widely considered to be the leading cause of ocular disease in cats and has been implicated in upper respiratory tract infections. Little, however is known about interstrain phylogenetic relationships, and details of the genomic structure. For the present study, twenty-six FHV-1 isolates from different cats in animal shelters were collected from eight separate locations in the USA, and the genomes sequenced. Genomic characterization of these isolates includied short sequence repeat (SSR) detection, with fewer SSRs detected, compared to herpes simplex viruses type 1 and 2. For phylogenetic and recombination analysis, 27 previously sequenced isolates of FHV-1 were combined with the 26 strains sequenced for the present study. The overall genomic interstrain genetic distance between all available isolates was 0.093%. Phylogenetic analysis identified four main FHV-1 clades primarily corresponding to geographical collection site. Recombination analysis suggested that interclade recombination has occurred.

Whole-Genome Sequences of Zika Virus FLR Strains after Passage in Vero or C6/36 Cells.

We report 26 complete genomes of Zika virus (ZIKV) isolated after passaging the Zika virus strain FLR in mosquito (C6/36) and mammalian (Vero) cell lines. The consensus ZIKV genomes we recovered show greater than 99% nucleotide identify with each other and with the FLR strain used as input.

Mosquito cell-derived West Nile virus replicon particles mimic arbovirus inoculum and have reduced spread in mice.

Half of the human population is at risk of infection by an arthropod-borne virus. Many of these arboviruses, such as West Nile, dengue, and Zika viruses, infect humans by way of a bite from an infected mosquito. This infectious inoculum is insect cell-derived giving the virus particles distinct qualities not present in secondary infectious virus particles produced by infected vertebrate host cells. The insect cell-derived particles differ in the glycosylation of virus structural proteins and the lipid content of the envelope, as well as their induction of cytokines. Thus, in order to accurately mimic the inoculum delivered by arthropods, arboviruses should be derived from arthropod cells. Previous studies have packaged replicon genome in mammalian cells to produce replicon particles, which undergo only one round of infection, but no studies exist packaging replicon particles in mosquito cells. Here we optimized the packaging of West Nile virus replicon genome in mosquito cells and produced replicon particles at high concentration, allowing us to mimic mosquito cell-derived viral inoculum. These particles were mature with similar genome equivalents-to-infectious units as full-length West Nile virus. We then compared the mosquito cell-derived particles to mammalian cell-derived particles in mice. Both replicon particles infected skin at the inoculation site and the draining lymph node by 3 hours post-inoculation. The mammalian cell-derived replicon particles spread from the site of inoculation to the spleen and contralateral lymph nodes significantly more than the particles derived from mosquito cells. This in vivo difference in spread of West Nile replicons in the inoculum demonstrates the importance of using arthropod cell-derived particles to model early events in arboviral infection and highlights the value of these novel arthropod cell-derived replicon particles for studying the earliest virus-host interactions for arboviruses.

Influenza virus recruits host protein kinase C to control assembly and activity of its replication machinery.

Influenza virus expresses transcripts early in infection and transitions towards genome replication at later time points. This process requires de novo assembly of the viral replication machinery, large ribonucleoprotein complexes (RNPs) composed of the viral polymerase, genomic RNA and oligomeric nucleoprotein (NP). Despite the central role of RNPs during infection, the factors dictating where and when they assemble are poorly understood. Here we demonstrate that human protein kinase C (PKC) family members regulate RNP assembly. Activated PKCδ interacts with the polymerase subunit PB2 and phospho-regulates NP oligomerization and RNP assembly during infection. Consistent with its role in regulating RNP assembly, knockout of PKCδ impairs virus infection by selectively disrupting genome replication. However, primary transcription from pre-formed RNPs deposited by infecting particles is unaffected. Thus, influenza virus exploits host PKCs to regulate RNP assembly, a step required for the transition from primary transcription to genome replication during the infectious cycle.

Enhanced early West Nile virus infection in young chickens infected by mosquito bite: effect of viral dose.

Mosquito transmission of arboviruses potentially affects the course of viral infection in the vertebrate host. Studies were performed to determine if viral infection differed in chickens infected with West Nile virus (WNV) by mosquito bite or needle inoculation. Mosquito-infected chickens exhibited levels of viremia and viral shedding that were up to 1,000 times higher at 6, 12, and 24 hours post-feeding (PF) compared with those inoculated with 10(3) PFU by needle. Follow-up studies were conducted to determine if enhanced early infection was due to a higher viral dose inoculated by mosquitoes. Needle inoculation with successively higher doses of WNV led to higher early viremia and viral shedding; a dose >or= 10(4) PFU by needle was required to attain the high early viremia observed in mosquito-infected chickens. Mosquitoes inoculated WNV at this level as estimated by feeding on a hanging drop of blood (mean: 10(2.5), range: 10(0.7)-10(4.6) PFU). These results indicate that enhanced early infection in mosquito-infected chickens may be explained by higher viral dose delivered by mosquitoes. On the other hand, chickens infected by multiple mosquitoes (N = 3-11) had viremic titers that were 25-50 times higher at 6 and 12 hours PF than in chickens infected by a single mosquito, suggesting that viral dose is not the only factor involved in enhanced early infection. The likelihood that enhanced early infection in mosquito-infected chickens is due to a higher viral dose inoculated by mosquitoes and/or other factors (saliva, inoculation location, or viral source) is discussed.