Increased pathogenicity of West Nile virus (WNV) by glycosylation of envelope protein and seroprevalence of WNV in wild birds in Far Eastern Russia

Abstract

In this review, we discuss the possibility that the glycosylation of West Nile (WN) virus E-protein may be associated with enhanced pathogenicity and higher replication of WN virus. The results indicate that E-protein glycosylation allows the virus to multiply in a heat-stable manner and therefore, has a critical role in enhanced viremic levels and virulence of WN virus in young-chick infection model. The effect of the glycosylation of the E protein on the pathogenicity of WN virus in young chicks was further investigated. The results indicate that glycosylation of the WN virus E protein is important for viral multiplication in peripheral organs and that it is associated with the strong pathogenicity of WN virus in birds. The micro-focus reduction neutralization test (FRNT) in which a large number of serum samples can be handled at once with a small volume (15 μL) of serum was useful for differential diagnosis between Japanese encephalitis and WN virus infections in infected chicks. Serological investigation was performed among wild birds in the Far Eastern region of Russia using the FRNT. Antibodies specific to WN virus were detected in 21 samples of resident and migratory birds out of 145 wild bird samples in the region.

Figure 1

Survival curves of young chicks subcutaneously inoculated with WN virus 6-LP (A) and 6-SP (B) variants. Two days old male chicks were inoculated with 10⁻¹ (○), 10⁰ (△), 10¹ (☐), 10² (●), 10³ (▲) and 10⁴ (■) PFU of 6-LP, and 10² (●), 10³ (▲), 10⁴ (■) and 10⁵ (×) PFU of 6-SP. Chicks were observed daily for health conditions. The number of chicks used was 5 for each variants.

Figure 2

Histopathological and immunohistochemical findings of the 6-LP infected young chicks. (A) Photomicrograph of marked necrosis of myocytes of heart from a young chick with WN virus infection. HE stain. (B) Myocytes of heart are positively stained for WN virus antigen.

Figure 3

Viremic levels of young chicks subcutaneously inoculated with WN virus variants. Young chicks were inoculated with WN virus variants, 6-LP (▲) and 6-SP (●) in experiment (A), and B-LP (△)and B-SP (○) in experiment (B). Two days old chicks were inoculated with 100 PFU of all variants (n = 4). The virus titers in sera were measured by plaque assay on BHK cells. Mean (±SD) titers are from triplicate cultures.

Figure 4

Virus titer of WN virus variants in Culex pipiens pallens. Seven days old female mosquitoes (n = 4) were inoculated intrathoracically with 100 PFU of all variants. The virus titers in mosquito bodies were measured by plaque assay on BHK cells. The virus titers of 6-LP (▲) and 6-SP (●) were shown in (A) and those of B-LP (△) and B-SP (○) were shown in (B).

Figure 5

Viral titers in the serum (A), hearts (B), spleens (C), kidneys (D), brains (E), and livers (F) of 2-day-old chicks infected with 6-LP (○) or 6-SP (◆). Chicks were infected with 10² PFU subcutaneously (s.c.) in the femoral region, and blood and tissues were collected at 2 and 6 days post infection (d.p.i.). Virus titers were determined by plaque assay using BHK-21 cells (n = 5 or 6). Individual and mean PFU values are represented by symbols and bars, respectively. When mean values were calculated, the titers of samples below the detection limit (10³ PFU/mL) were considered to be 3.0. Error bars indicate standard deviations. p-values were calculated using unpaired Student t-tests.

Figure 6

Primary neutralizing antibody responses in chicks inoculated with WNV 6-LP or 6-SP. Two-day-old (A) and 3-week-old (B) chicks (n = 3–6) were inoculated with 10² PFU of WNV 6-LP (○) or 6-SP (◆). WNV neutralizing antibody titers were then measured by PRNT₈₀. Individual and mean PRNT₈₀ titers are represented by symbols and bars, respectively. When mean values were calculated, the titers of samples under the detection limit were considered to be 20.

Figure 7

Cytokine and transcription factor mRNA levels in the hearts of 2-day-old chicks inoculated with WNV 6-LP or 6-SP. Chicks were infected with 10² PFU of virus administered subcutaneously in the femoral region, and tissues were collected at 2 and 6 d.p.i. Total RNA was then extracted and cDNA synthesized. SYBR Green-based quantitative real-time PCR was performed using the synthesized cDNA. Relative quantification of cytokine gene expression was done using the CT method. The CT data for each cytokine were normalized against the b-actin levels in the same sample. * and ** indicate statistically significant differences (* p, 0.01; ** p, 0.05) in cytokine and transcription factor mRNA levels compared with mock-infected chicks.

Figure 8

Cytokine and transcription factor mRNA levels in the spleens of 2-day-old chicks inoculated with WNV 6-LP or 6-SP. Chicks were infected with 10² PFU of virus administered subcutaneously in the femoral region, and tissues were collected at 2 and 6 days post infection (d.p.i.).Total RNA was then extracted and cDNA synthesized. SYBR Green-based quantitative real-time PCR was performed using the synthesized cDNA. Relative quantification of cytokine gene expression was done using the CT method. The CT data for each cytokine were normalized against the b-actin levels in the same sample. * and ** indicate statistically significant differences (* p, 0.01; ** p, 0.05) in cytokine and transcription factor mRNA levels compared with mock-infected chicks.

Figure 9

Cytokine and transcription factor mRNA levels in the spleens of 3-week-old chicks inoculated with WNV 6-LP or 6-SP. Chicks were infected with 10² PFU subcutaneously (s.c.) in the femoral region, and tissues were collected at 2 and 6 days post infection (d.p.i.). Total RNA was extracted and cDNA synthesized. SYBR Green-based quantitative real-time PCR was performed using the synthesized cDNA. Relative quantification of cytokine gene expression was done using the CT method. The CT data for each cytokine were normalized against the b-actin levels in the same sample. * Statistically significant differences (p < 0.01) in cytokine and transcription factor mRNA levels compared with mock-infected chicks.

Figure 10

Primary neutralizing antibody responses in chicks inoculated with Japanese encephalitis (JE) and West Nile (WN) viruses. Two-day-old chicks (n = 4) were inoculated with 100 plaque forming units (PFU) of (A) JE virus and (B) WN virus, and 3-week-old chicks (n = 4) were inoculated with 1,000 PFU of (C) JE virus and (D) WN virus. Anti-JE virus (◇) and -WN virus (●) neutralizing antibody titers were measured by FRNT₈₀ and are expressed as the mean ± SD.

Figure 11

Neutralizing antibody responses in chicks after a secondary challenge with heterologous viruses. Two-day-old chicks (n = 4) were inoculated with 100 plaque forming units (PFU) of primary viruses: (A) Japanese encephalitis (JE) virus, (B) West Nile (WN) virus. After 3 weeks, the chicks (23 days old) were inoculated againwith 1,000 PFU of heterologous virus: (A) WN virus, (B) JE virus. Anti-JE virus (◇) and -WN virus (●) neutralizing antibody titers were measuredby FRNT₈₀ and are expressed as the mean ± SD.