New strains of Japanese encephalitis virus circulating in Shanghai, China after a ten-year hiatus in local mosquito surveillance

Abstract

Background: Continuous vector pathogen surveillance is essential for preventing outbreaks of mosquito-borne diseases. Several mosquito species acting as vectors of Japanese encephalitis virus (JEV), dengue virus, Zika virus, malaria parasites and other pathogens are primary mosquito species in Shanghai, China. However, few surveys of human pathogenic arboviruses in mosquitoes in Shanghai have been reported in the last ten years. Therefore, in this study, we evaluated mosquito activity in Shanghai, China during 2016 and tested for the presence of alphaviruses, flaviviruses, orthobunyaviruses and several parasitic pathogens.

Results: Five pooled samples were JEV-positive [4/255 pools of Culex tritaeniorhynchus and 1/256 pools of Cx. pipiens (s.l.)] based on analysis of the NS5 gene. Alphaviruses, orthobunyaviruses, Plasmodium and filariasis were not found in this study. Phylogenetic and molecular analyses revealed that the JEV strains belonged to genotype I. Moreover, newly detected Shanghai JEV strains were genetically close to previously isolated Shandong strains responsible for transmission during the 2013 Japanese encephalitis (JE) outbreak in Shandong Province, China but were more distantly related to other Shanghai strains detected in the early 2000s. The E proteins of the newly detected Shanghai JEV strains differed from that in the live attenuated vaccine SA14-14-2-derived strain at six amino residues: E130 (Ile→Val), E222 (Ala→Ser), E327 (Ser→Thr), E366 (Arg→Ser/Pro), E393 (Asn→Ser) and E433 (Val→Ile). However, no differences were observed in key amino acid sites related to antigenicity. Minimum JEV infection rates were 1.01 and 0.65 per 1000 Cx. tritaeniorhynchus and Cx. pipiens (s.l.), respectively.

Conclusions: Five new Shanghai JEV genotype I strains, detected after a ten-year hiatus in local mosquito surveillance, were genetically close to strains involved in the 2013 Shandong JE outbreak. Because JEV is still circulating, vaccination in children should be extensively and continuously promoted. Moreover, JEV mosquito surveillance programmes should document the genotype variation, intensity and distribution of circulating viruses for use in the development and implementation of disease prevention and control strategies.

Keywords: Culex pipiens; Culex tritaeniorhynchus; Japanese encephalitis; Mosquito-borne diseases; SA14-14-2.

Fig. 1

Map of survey sites for the mosquito-borne pathogen surveillance programme in 2016 in Shanghai, China. Squares represent sites using the CO₂-baited trap, triangles represent sites using the labor hour method and color-filled symbols represent Japanese encephalitis virus detection. Abbreviations: HP, Huangpu District; XH, Xuhui District; CN, Changning District; JA, Jing’an District; PT, Putuo District; HK, Hongkou District; YP, Yangpu District

Fig. 2

Maximum likelihood phylogenetic tree of partial NS5 gene sequences of flavivirus. The maximum likelihood tree was constructed by the GTR + I + G model. The GenBank accession number, virus name, origin, and country/province are noted. The JEV sequences obtained in this study are marked in red. The numbers above each branch represent the bootstrap support for the maximum likelihood, neighbor-joining, and Bayesian analyses, respectively, based on 1000 replicates. The scale-bar indicates 0.1 substitutions per site. Sequences shaded tan represent mosquito-borne flavivirus, those shaded sky blue represent tick-borne flavivirus, those shaded aquamarine represent no-known vector flavivirus, and those shaded khaki represent insect-specific flavivirus. Abbreviations: JEV, Japanese encephalitis virus; SLEV, Santa Louis encephalitis virus; TBEV, tick-borne encephalitis virus; MMLV, Montana myotis leukoencephalitis virus; MODV, Modoc virus; CxFV, Culex flavivirus; QBV, Quang Binh flavivirus; NAKV, Nakiwogo virus; AEFV, Aedes flavivirus; KRV, Kamiti River virus; CFAV, cell fusing agent virus

Fig. 3

Maximum likelihood phylogenetic analysis of Japanese encephalitis virus pre-membrane gene sequences. The maximum likelihood tree was constructed under the TrN + G model. The GenBank accession number, origin, country/province and genotype of each strain are noted. The JEV sequences obtained in this study are marked in red. The numbers above each branch represent the bootstrap support of the maximum likelihood, neighbor-joining, and Bayesian analyses, respectively, based on 1000 replicates. The scale-bar indicates 0.05 substitutions per site. Sequences shaded tan represent the GI-a genotype, those shaded rose-brown represent the GI-b genotype, those shaded sky blue represent the GII genotype, those shaded khaki represent the GIII genotype, those shaded aquamarine represent the GIV genotype, and those shaded thistle represent the GV genotype

Fig. 4

Maximum likelihood phylogenetic analysis of Japanese encephalitis virus envelope gene sequences. The maximum likelihood tree was constructed under the GTR + I + G model. The GenBank accession number, origin, country/province and genotype of each strain are noted. The JEV sequences obtained in this study are marked in red. The numbers above each branch represent the bootstrap support of the maximum likelihood, neighbor-joining, and Bayesian analyses, respectively, based on 1000 replicates. The scale-bar indicates 0.05 substitutions per site. Sequences shaded tan represent the GI-a genotype, those shaded rose-brown represent the GI-b genotype, those shaded sky blue represent the GII genotype, those shaded khaki represent the GIII genotype, those shaded aquamarine represent the GIV genotype, and those shaded thistle represent the GV genotype

Fig. 5

Sequence comparison of amino acid differences in the envelope protein of Japanese encephalitis virus (JEV). Sequence comparisons were performed among the live attenuated vaccine SA14-14-2, SA14, and newly detected Shanghai JEV strains (marked in red), as well as strains suspected to have contributed to prior Japanese encephalitis outbreaks (2013 in Shandong Province, China; 2010 in Korea; and 2006 in Wuhan Province, China) near Shanghai. Dots indicate consensus. The GenBank accession numbers and countries/provinces are noted. Triangles represent eight amino acids (F107L, K138E, V176I, A177T, H264Q, M279K, V315A, and R439K) related to virus attenuation; circles represent two pairs of co-evolving sites (residues S89N to F360Y and M129T to I141V) observed in GI; squares represent four sites (E123, E209, E327, and E408) in the E protein used for haplotype definitions; stars represent the amino acids (I130V, A222S, S327T, R366S/P, N393S, and V433I) of newly detected Shanghai JEV strains that were not consistent with those in the SA14 strain

Fig. 6

Genetic distances of the envelope genes from five genotypes of Japanese encephalitis virus (JEV). Genetic distance analyses were performed using Kimura’s two-parameter model based on 69 JEV envelope sequences from GenBank and two sequences obtained in this study. The GenBank accession numbers of involved sequences are available in Fig. 4. Y-axis, genetic divergence; X-axis, JEV genotypes