This study describes the epidemiological and genetic investigation of CCHF viruses detected in serum samples of clinically suspected and subsequently laboratory confirmed patients, received during September to October, 2008. The study subjects include patients from Pakistan and Afghanistan that were admitted to Fatima Jinnah General and Chest Hospital, Quetta.
In Pakistan, CCHFV is endemic to many areas of Baluchistan that harbor ticks and appropriate vertebrate hosts which remain in close contact with humans. Geographically, Baluchistan is the largest province of Pakistan and occupies area of 348,189 km2 with hyper-arid climate. Livestock farming is the main work practice and source of income for majority of the population. 93% of the provincial land area comprises rangelands that cannot sustain vegetation throughout the year, leading to seasonal migrations of nomadic populations to areas within the Baluchistan province and bordering areas of Afghanistan along with their livestock in search of food and water for themselves as well as their cattle and sheep flocks. These animals may serve as disease carrier or carry CCHFV infected ticks and become a major source of transmission across their migratory places.
Although the CCHF infection has been reported in Pakistan since 1976, the available data on CCHF cases during the past few years is very limited. It is most likely that many of cases have not been reported due to lack of proper surveillance system in Pakistan. This concern has been raised by previous studies [12, 16] highlighting an unremitting need of surveillance, laboratory and medical facilities, especially in the CCHF endemic areas. This insufficiency in health services has led to alternative diagnostic measures based on clinical presentations and disease manifestations in patients. Although clinical diagnosis is useful, the high mortality of CCHF and propensity for nosocomial transmission warrant rapid laboratory confirmation to control and minimize epidemics.
Hyalomma tick species are considered as the main transmission vector in European as well as Asian and African countries especially H. marginatum marginatum and H. analiticum analiticum however, there have been reports of other tick species found positive for CCHF viruses like Ornithodoros lahorensis, Rhipicephalus sanguineus, Rhipicephalus bursa, and dermacenter species . For instance, CCHF virus was isolated from Boophilus ticks in Madagascar, while a Greek isolate of the virus was recovered from Rhipicephalus. In admonition to the role of ticks, there have been findings that CCHF infections usually occur in geographies with hot climate, where land farming is common. Many cases are reported from areas with increased animal farming where agricultural workers regularly come into contact with livestock . It is likely that in endemic areas, infected livestock that notably remain asymptomatic may serve as virus reservoirs. In Kosovo, 32.6% while in Azerbaijan Shargi province of Iran, 25-80% of the sheep population was found seropositive during 1975–1999 survey for CCHF respectively . This necessitates the importance of generating information on the prevailing tick vectors in our country and their disease association to CCHF infections. Similarly, most of the cases in Turkey during 2005 were either the result of tick-bite or patients who had viral exposure through livestock handling . To date serological surveys of this nature have not been possible in Pakistan, although CCHF cases reported in Karachi city were linked to farming practices with sheep and goats brought from Baluchistan to Sindh province . Similarly, large scale epidemiological surveys are required to study tick fauna in the context of CCHF infection. This will be important in defining hotspots of disease and constructing risk factors associated with particular regions. Such information will also help to define the range of tick species capable of hosting and transmitting CCHFV to humans, ultimately building evidence for vector and disease control programs that can directly support public health campaigns for populations who live in endemic areas.
Genetic sequencing of CCHFV genome provides knowledge of virus lineages which is a useful molecular epidemiological information and helps to understand risk areas and the transmission patterns of viruses , in addition to the long range relationships between outbreaks of disease. The genetic information of CCHF viruses reported from Pakistan is very limited due to lack of laboratory facilitates, however, the information available to date represents data of samples collected intermittently during 1976, 2000, 2001 and 2002 which were sent to international laboratories i.e. South Africa (National Institute for Communicable Diseases, Sandringham) and US-Centers for Disease Control and Prevention. Genetic characterization of all such viruses revealed that they are phylogeneticaly clustered with the viruses from neighboring Middle East Asian countries such as Iran, Afghanistan and UAE when their partial S-gene segments are compared . Beyond the year 2002, no genetic information of CCHF viruses have been available despite the increased number of cases every year . Our findings presented here further substantiate the existence of Asia-1 genotype in Pakistan explaining its vast endemicity in this region.
The recent report of a CCHF virus (strain; AFG09-2990) from Afghanistan showing 100% S-segment identity with the Pakistani strains  provides support for the close transmission links between these neighboring countries. It has been reported that Iranian strains also hold close similarity to Pak-Matin strain (Accession No. U75678) in Pakistan, indicating shared transmission sources between the two countries . Likewise, another study analyzed the random interchange of CCHFV sequences between countries and found that Asia-1 genotype moves freely between Pakistan and Iran . Tahmasebi et al. support such assumptions by finding that all CCHF viruses from Hamadan province of Iran clustered together with the CCHFV strains of Pakistan [17, 19] proving evidence that the unchecked and uncontrolled animal movements between these countries remain a source of sustained and constant introductions of CCHFV to naive populations . In the same way, animal trade between Pakistan and Middle Eastern countries also provide sources for the importation of CCHF virus from Pakistan to other countries, including UAE as evidenced by an outbreak in Dubai and was found linked to the transportation of cattle from Pakistan . The Madagascar CCHFV strain isolated from Boophilus microplus species is believed to be introduced through cattle imported from Asia as these tick species occur primarily in Pakistan and India . Two Chinese isolates (strain Fub900009 and C-68031) of CCHF virus have shown close homology with the viruses from Pakistan indicating frequent livestock trading or cultural and economic exchanges [27, 28]. The presence of viruses with similar genetic make-up but in different geographical areas indicates the movement of CCHF virus-infected livestock or uninfected livestock carrying infected ticks due to considerable movement of sheep and goats from Pakistan into the Arabian Peninsula particularly at the time of religious festivals . All such reports highlight that the CCHFV is not only contained within specific areas of Pakistan but has contributed to the transmission of virus across borders especially to the geographically neighboring countries . Likewise, unlawful animal trade and uncontrolled population movements occur between Pakistan and Afghanistan through Quetta city due to the very similar cultural and tribal civilizations inherent across the bordering areas of both countries. Baluchistan province is a major source of animal’s skin and hides production, and also serves as a corridor for receiving skins and hides from Southern Afghanistan and Southeastern parts of Iranian Baluchistan via the Taftan gateway .
These findings provide significant evidence of frequent animal trade and movements and call for a dire need of strict quarantine regulations to be devised to ensure CCHFV screening of all animals to be transported from Pakistan to other world regions. More detailed tracing of animal movements, their role as potential disease carriers along with the analysis of virus genome sequences should be undertaken. This will provide beneficial outcomes due to the fact that assessment of livestock antibodies against CCHFV is one of the best indicators of CCHF risk to humans as well as effectively contribute to monitor the risk of CCHF in non-endemic regions . Similarly, due to importance of migration patterns of CCHFV, more resources must be allocated for improved surveillance, sampling and collecting genetic information through sequencing of CCHFV strains .
Our study limitations include the genetic analysis based on partial S-gene sequence due to very low quantity of sample and insufficient resources, however, it can correctly define the genetic classification of CCHF viruses into seven distinct genotypes as utilized by previous studies [1, 14, 30, 31].