| An infectious disease emerged in2009in rural areas of Hubei, Henan and Shandong provinces.The disease was characterized by fever, thrombocytopenia, leukocytopenia and gastrointestinal symptoms with a case fatality rate of approximately10%-30%. A novel virus, designated SFTS bunyavirus, was isolated from patients who presented with fever, thrombocytopenia, leukocytopenia, and multiorgan dysfunction in2010. RNA sequence analysis revealed that the virus was a new member of the genus phlebovirus in the Bunyaviridae family.Members of the family Bunyaviridae were discovered worldwide, and some of them were significant pathogens in human, animals and plants. Most of bunyaviruses were arbovirus and spread through the vertebrate hosts and hematophagous arthopods, such as mosquitos, phlebotomine flies, and ticks. Viruses in the genus Hantavirus were unique among the Bunyaviridae in that they did not infect insect vectors. In addition, an alternative route of nonvectored transmission had been reported for some nairoviruses and phleboviruses, including Crimean-Congo hemorrhagic fever virus and Rift Valley fever virus. These viruses could be spread by exposure to infected human and animal tissues or body fluids [2]. The model of transmission cycle of SFTSV in nature was still unclear.So far, the confirmed SFTS cases had been found in15provinces of China. Since2008, the accumulative total of SFTS cases and dead cases were1531and101, of which in Shandong province were370and42, with the proportion of24.2%and41.6%respectively. Yantai city of Shandong province, as the mainly epidemic area, its SFTS cases were44and81in2010and2011, account for39.2%and48.2%of Shandong province, respectively. SFTS cases were presented as highly sporadic and mainly located in the rural fields of the mountainous and hilly areas. This disease mostly occurred in the spring and summer, slightly differences may exist among different regions. Human were generally susceptible to this novel virus, living or working in the hills, mountains and forests, as well as tourists going to such areas had higher risk of infection. Most of cases were old people, and there was no difference between male and female. The transmission of SFTSV was still unknown, while tick biting had been reported in part of cases. In addition, there were recently reported that exposure to the acute phase blood of critically ill patients may get the possibility of human to human transmission, more attention and personal protection should be considered by family members of patients and health care workers [3].In order to understand the potential vectors and hosts, we selected the rural area of Laizhou and Penglai of Shandong province which had SFTS confirmed case before2010to carry out the investigation from April to November2011. Serum samples such as sheep, cattle, dogs, pigs and chickens were collected from affected area monthly, and to detest viral nucleic acid and antibody by real time RT-PCR and double antigen sandwich ELSA, then to analysis the infection situation of SFTSV in vectors and animals. At the same time, engorged ticks were collected from common domesticated animal species include sheep, cattle, dogs, and chickens. Field ticks were sampled by "the woolen flannel cloth dragging method". All ticks were crushed and tested by real time RT-PCR. We tried to isolate SFTSV strain and amplified S segment sequences from viral RNA positive samples. Through homology comparison and phylogenetic analysis and the experiments described before, we attempted to know the etiology and molecular characteristics of SFTSV, elaborate the likelihood of domestic animal and ticks as the hosts and vectors, carry out relevant research on biological characteristics of the SFTSV and provide a theoretical basis for the viral disease prevention.The dissertation was divided into three parts:1. Seroepidemiological survey of domestic animals in Laizhou and Penglai rural area of yantai city, Shandong province from April to November2011.Animals of sheep, cattle, dogs, pigs, chickens were sampled monthly in the villages where SFTS human cases were reported in2010.50to100animals of each species were sampled each month from April to November of2011(appendix3) and the sera were collected for detection of viral RNA, antibodies and virus isolation. Each sampled animal was marked with a unique label except pigs, which were sampled at the time of slaughter. Sampled animals were excluded in subsequent sampling events. In addition, to monitor new infections of SFTSV, a cohort of38sheep (LZ, n=17; PL, n=21) negative for SFTSV viral RNA and antibodies was established, and the animals were sampled every10days between June20th and November30th. Sera were aliquoted and kept in cryovials in liquid nitrogen. Viral RNA and antibodies were first screened at the local CDC laboratories in LZ and PL, respectively, and the results were confirmed later in the national laboratory of China CDC.To investigate the infections of SFTSV in domesticated animals, we sampled a total of3039animals in two counties from April to November2011. SFTSV-specific antibodies were detected in328of472sheep (69.5%),509of842cattle (60.4%),136of359dogs (37.9%),26of839pigs (3.1%), and250of527chickens (47.4%). SFTSV RNA was detected in all sampled animal species, but the prevalence was low, ranging from1.7%to5.3%. A cohort study in38sheep was done to determine the time course of seroconversion to SFTSV. SFTS viruses were isolated from sheep, cattle, and dogs, and shared over95%sequence homology with human isolates from the same endemic regions. These findings demonstrate that natural infections of SFTSV occur in several domesticated animal hosts in endemic areas, and that the virus has a wide host range.2. Seroepidemiological survey of healthy population in Laizhou and Penglai rural area of yantai city, Shandong province from April to November2011.The investigation of SFTSV seroepidemiology was conducted during April in thirty rural villages in which several human infections have been reported over the past two years in northern yantai city with a total registered population of31,000. Multi-stage stratified random sampling was adopted and2,590healthy peoples’ blood samples were collected from volunteers of these villages at last. The number consisted of a portion (8.3%) of the population in these villages. A standardized questionnaire was used to obtain information on age, gender, history of illness and occupation of each participant. Every sample was selected in accordance with the principle of random sampling. All of them were assembled into the following age strata:0-10,11-20,21-30,31-40,41-50,51-60and60-. Of the2590analyzed sera,5.4%(140) were positive for SFTSV IgG. There were no statistically significant difference in gender and age.(P>0.05). But there was statistically significant difference in occupation (P＜0.05).3. Survey of ticks in Laizhou and Penglai rural area of Yantai city, Shandong province.Ticks were sampled in the villages where SFTS human cases were reported in2010. All ticks were kept alive in collecting tube at room temperature for one week until storage in-80℃, which aimed to make the engorged ticks digestion of the blood. After species identification all ticks were grouped into2,044pools according to species, host, and geographic origin.To determine tick carrying SFTSV, viral RNA was extracted from each tick pools separately. Then total RNA was extracted from tick grinding fluid and detected for viral RNA and virus isolation. In this study, a total of3,145ticks were collected including2,251engorged ticks from various host species and894field ticks from grassland. Among all ticks H. longicornis Rhinpicephalus microplus, Haemaphysalis campanulata and Dermacentor sinicus were found according to morphological examination and H. longicomis ticks were the dominant species (97%).The results showed that126pools out of3145ticks (MIR=4%) were SFTSV positive and11full S segment sequences were successfully amplified from ticks. |
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