Mosquitoes And Mosquito-borne Arboviruses And The Relation With Local Human Disease In Qinghai Province

BackgroundQinghai Province (short for "Qinghai") is located in the western region of main-land China, and most of its area is located in the Qinghai-Tibet Plateau. The average altitude of Qinghai is over 3 000m. Qinghai is an inland province, and borders Gansu Province on the northeast, Xinjiang Uygur Autonomous Region (short for "Xinjiang") on the northwest, Sichuan Province on the southeast, and Tibet Autonomous Region (short for "Tibet") on the southwest. Qinghai has a vast area about 72 million square kilometers, which result complex geographical landscape, and more categories of vector and vector-borne diseases. Previous researches showed that bacterial vector-borne diseases such as plague, typhus fever, brucellosis, and Lyme disease have occurred in Qinghai.Yet, no systematic investigation on mosquito-borne arboviruses has been carried out in Qinghai. However, seasonal febrile cases of unknown etiology are frequently occurred in local, which would be caused by viruses. Japanese encephalitis (short for "JE") is an important mosquito-borne arboviral disease. Since the 1950s, there was no JE case reported in Qinghai, therefore Qinghai has been considered to be non-endemic area of JE. However, Gansu and Sichuan, the adjacent provinces of Qinghai, have reported JE cases yearly, so it was posobile that there may be mosquito-borne arboviruses such as JEV in Qinghai.In recent years, Qinghai has become a crucial region that connects inland provinces of China with countries in South Asia such as Nepal, India, and Bangladesh, along with communication and trade among Tibet and the other western provinces in China has been intensified as a result of the construction of the Qingzang Railway which connects Xining city (capital of Qinghai) to Lhasa city (capital of Tibet). Consequently, trade and travel across Qinghai are increasing rapidly, the likelihood of introduction or exportation of vectors and vector-borne diseases increases concomitantly.Therefore, it is important to strengthen the detection and monitoring of vector-borne diseases, especially mosquito-borne arboviruses in Qinghai, which would help us to cmplete information about arboviruses in western plateau, as well as to controll and prevent mosquito-borne arboviral diseases in Qinghai, further in China.ObjectiveTo understand the distribution of mosquitoes and mosquito-borne arboviruses in Qinghai, and focus on newly-isolated mosquito-borne arboviruses infection in local residenta and livestock, and further definitude clinical and epidemical features of related diseases. The data will provide background information for prevention and control of mosquito-borne arboviral diseasea in local, and even mainland of China.MethodsMosquitoes, livestock serum, healthy residents serum and febrile patients serum samples were collected from Qinghai in summer of 2007～2009. Specimens were stored at liquid nitrogen or -80℃refrigeratory in the local center for disease control and prevention (CDC), and then shipped to the Beijing laboratory in dry ice.Mosquitoes were homogenized and centrifuged, then the supernatants were inoculated into monolayers of BHK-21 and C6/36 cells to isolate virus. The virus isolates were identified by serology and molecular biology systematically. Reverse transcription polymerase chain reaction (RT-PCR) was conducted with primers designed for special genuses of arboviruses, and positive DNA fragments were extracted and sequenced by a commercial provider. The analysis on nucleotide sequences and derived amino acid sequences and further phylogenetic analysis were conducted using software of ClustalX1.83, MegAlign, Mega4 and Genedoc3.2.IFA was applied to detect immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies agaist newly-isolated arboviruses in serum samples collected from healthy residents and local livestock. Seropositive samples by IFA were tested for neutralizing antibodies by 90% plaque-reduction neutralization tests. Further analysis was made for distribution characteristic gender, region and age among seropositive samples.Acute phase serum samples from febrile patients were tested for IgM antibodies against newly-isolated arboviruses. For patients in whom IgM antibodies were detected in acute phase sera, paired serum samples were taken in the convalescent phase, usually 21 days after onset, and followed up regulal for 3 months. Plaque-reduction neutralization test was progressed to confirm whether neutrolizing antibodies presented 4-fold or greater increase or not. Serum samples were examined for viral nucleotides by nested PCR. For PCR-positive serum samples, suckling mice were applied to isolate viruses.Results1. Distrubition of mosquitoes. Mosquito samples were collected from four regions in Qinghai, including Geermu city, Xining city, Minhe county, and Gangcha county in 2007～2009. A Total of 23 608 mosquitoes representing ten species from three genera (Aedes, Culex, and Anopheles) were collected, including Ae. (Och.) detritus, Ae. (Och.) dorsalis, Ae. (Och.) flavidorsalis, Ae. (Och.) caspius, Ae. (Och.) pullatus, Aedes vexans, Aedes not identificated, Culex modestus, Culex pipiens pallens, Anopheles nigerrimus. The predominant species varied by regions with Ae. (Och.) detritus being the most abundant species in Geermu city (29.32%,2523/8 604), Ae. vexans in Xining City (84.47%,4 225/5 002), Cx. modestus in Minhe county (32.80%,2 448/7 463), and Ae. (Och.) flavidorsalis in Gangcha county (98.03%, 2489/2539). This study represents the first report of Anopheles nigerrimus in so far north in China. It is important to note that no Cx. tritaeniorhynchus was collected in Qinghai, which is the major vector of JEV in China. The absence of Cx. tritaeniorhynchus is consistent with the absence of Japanese Encephalitis cases in Qinghai.2. Isolation and identification of mosquito-borne arboviruses. All mosquito specimens were packaged into 479 pools, and were homogenized and centrifuged, then the supernatants were inoculated into monolayers of BHK-21 and C6/36 cells to isolate virus. A specimen was regarded as a positive isolate if it caused CPE in three successive cell passages, and a total of 19 virus isolates were obtained. Both QH07029 and QH07060 reacted with immune ascites against Bunyaviruses and against TAHV prototype Bardos 92 strain, and QH07130 reacted with immune ascites against LNV. Molecular biological identification showed that the 19 virus isolates were TAHV (2 strains), LNV (1 strains), Orbivirus (2 strains), and Culex pipiens pallens Densovirus (CppDNV,14 strains). Molecular phylogenetic analysis showed TAHV Qinghai isolates located in a separate sub-branch of TAHV, and were independent of European isolates and Xinjiang isolates. As TAHV is an important agent belong to Bunyaviridae, and respect to publichealth across Europe, and was recently isolated in China, so this study will focus on TAHV, especially on prevalence in local and relation with local disease.3. Biologic and molecular characters of TAHV. Two virus strains with obviously different plaque morphology were derived from Qinghai TAHV QH07060 strain by plaque purification, which were denominated as QH07060-LP (large plaque) and QH07060-SP (small plaque) respectively. The CPE of QH07060-LP in BHK-21 cells presented 6 hours ealier than QH07060-SP. The reproduction capacity of QH07060-LP in BHK-21 cells was 100～1 000 folds higher than QH07060-SP. Moreover, the neurovirulence of QH07060-LP and QH07060-SP in suckling mice was significantly different, with the lethal time of QH07060-LP and QH07060-SP virus was 4 days and 6 days respectively. The whole genome of QH07060-LP and QH07060-SP were both 12 260nt, and nine nucleotide differences were found, of which one was located in the 5'-UTR of S segment, and the other eight in protein-coding region. These nine different nucleotides resulted in 6 amino acid difference. For these six amino acid difference spot were conservative in other other TAHV as well as California complex seragroup viruses, which prompts the mutation of these sits may be the molecular foundation of differrent phenotype and biology of QH07060-LP and QH07060-SP.4. Prevalence of TAHV antibodies in local residents. A total of 1 078 serum samples of local residents were collected from Geermu city, Xining city, and Minhe county from August to September in 2007 and 2008. Of 1 078 serum samples,19 were IgG positive (1.76%),23 were neutrolizing antibodies positive (2.13%). The positive rate in Geermu city was greater than both in Xining city and Minhe county, and showed an obvious characteristic of geographical distribution among these three regions. All of the seropositive samples came from the under 30 age groups. The highest seroprevalence of neutrolizing antibodies was found in the 20～29 age groups (4.35%). Combined the fact that TAHVs were isolated and TAHV antibodies were detected in Qinghai, TAHV infection would occur in Qinghai, especially in Geermu city.5. Prevalence of TAHV antibodies in local livestock. A total of 240 serum samples from local livestock were collected in Geermu city, Xining city, and Minhe county from August to September in 2008. Species included cow, sheep, and swine. Of 90 cow sera,3 were IgM positive, and 6 were IgG positive. Of 90 sheep sera,7 were IgM positive, and 9 were IgG positive. Of 60 swine sera,3 were IgM positive, and 2 were IgG positive. Most of the positive livestock sera were collected from Geermu city where 13.7% (5/30) of cows and 26.7% (8/30) of sheep were positive. There were also positive sera collected from Xining city and Minhe county, but positive rates in both locations were lower than in Geermu city. In 2009, serum samples were collected from local livestock in Geermu city, and spieces were expanded to cow, sheep, swine, rabbits, chickens, and ducks. The overall seroprevalence of IgG antibodies was 11.5% (42/364), and the highest was found in rabbit (38.0%,5/13), suggesting that the rabbit may play an important role in the natural cycle of the TAHV in local.6. Disease caused by TAHV in Qinghai. Research in foreign revealed that human infection TAHV manifested varying degrees of fever, therefore an investigation was progressed in febrile patients in Geermu city in, Xining city and Minhe county in Qinghai from July to September,2009. To determine whether disease caused by TAHV occured or not in local, especial investigation was conducted in Geermu city A total of 6 villages were involved, covering an area of approximately 240 square kilometers. During this investigation, ward inspections were conducted in rural hospitals and clinical information and specimens were collected from patients who mainly complained of acute fever. Laboratory tests showed that TAHV IgM antibodies were detected from acute sera of febrile patients, with total prevalence of 3.17% (21/661); paired sera presented 4-fold or greater increase of TAHV neutralizing antibody titers, and TAHV nucleotides were detected in acute sera. Clinical features of TAHV infection cases commonly included fever, malaise, headache, respiratory symptoms, and gastrointestinal disorders. Follow-up surveys revealed that all patients recovered in 2-5 days after onset and no serious cases or mortality was observed after follow-up survey of 3 months.ConclusionThroughout a three-year study in Qinghai, the distribution of mosquitoes were discribed definitely, and the difference on mosquito species distribution is associated with local physical geography; 19 strains of arboviruses were obstained from mosquitoes, including TAHV, LNV, Orbivirus and CppDNV, which were first isolated in the Qinghai-Tibet Plateau. TAHV antibodies were detected in local residents and livestock. Seroprevelence of TAHV antibodies were higher in Geermu city than both in Xining city and Minhe county. Furthermore, TAHV infection cases were confirmed by laboratory tests in febrile patients in Geermu city of Qinghai. It is the first time TAHV infection was confirmed in China, which is the third known mosquito-borne arboviral disease besides JE and Dengue.