Chikungunya Outbreak Confined To Bagan Panchor Village

CAkidd · Apr 4, 2006

April 04, 2006 19:47 PM

Chikungunya Outbreak Confined To Bagan Panchor Village

Datuk Dr Chua Soi Lek

KUALA LUMPUR, April 4 (Bernama) -- Health Minister Datuk Dr Chua Soi Lek, Tuesday said the outbreak of rare mosquito-borne disease, Chikungunya, has so far only been detected in Bagan Panchor village in Pantai Remis, Perak and has not spread to any other places in the country.

"It's still confined there, I hope it would be contained," he said.

Speaking to reporters at the sideline of the MCA leaders dialogue session with Chinese community leaders in Bukit Kiara, Dr Chua said the ministry had deployed two top experts -- virologist Dr Chua Kaw Beng and physician Dr Christopher Lee, to help the relevant authorities to contain the disease even it was not fatal.

Dr Chua Kaw Beng is a renowned virologist who has detected the Chikungunya outbreak in Port Klang six years ago where 27 people were infected with the disease. Chikungunya was first described in Tanzania, Africa in 1952.

Dr Chua said several measures to contain the outbreak, believed to be brought in by foreign workers, working in the area had been implemented.

A team has been sent to work with the local authorities there to clean up the villages as the area has been found to have very high aedes breeding index (27 per cent) compared to other areas where the index would be normally around one per cent.

Other measures included setting up a mobile clinic in the village to provide treatment for those infected while the serious cases would be transferred to Taiping Hospital, he said, adding that the ministry hoped to contain the outbreak in the next 10 days.

So far, 200 villages have been infected with the Chikungunya with five of them warded at Taiping Hospital. However, four of them had been discharged yesterday, leaving only one patient still being hospitalised.

Dr Chua also expressed disappointment with private clinics there for not reporting the outbreak of Chikungunya much earlier to the ministry.

He said the ministry was told that the outbreak had occurred much earlier -- after the Chinese New Year but none of the cases had been reported to the Health department.

"We have never been alerted until March 26 when one GP (General Practitioner) alerted us that he saw a lot of cases on the same day.

To the private doctors, "when they see a lot of patients with the same complaint, they should alert us that there is in-fact an epidemic. They should not keep quiet," he said.[/b

Dr Chua said if the Chikungunya outbreak was not detected early, it could contribute to the massive outbreak as recorded on the French island of Re'union in the Indian Ocean where about 157,000 residents, of the total population of 777,000 have been hit by the virus.

The name Chikungunya is derived from Swahili "that which bends up", referring to stooped posture developed as a result of arthritic symptoms of the disease. The symptoms included fever, rashes, arthritis affecting multiple joints and headache.

-- BERNAMA

http://www.bernama.com/bernama/v3/news.php?id=190032

DB · Apr 4, 2006

Re: Chikungunya Outbreak Confined To Bagan Panchor Village

1) There are over 230,000 cases in La Reunion.
2) Every attempt at containing Chikungunya has failed. Malaysia will be no different.
3) It is not being spread via mosquitoes so public health officials should be advising people implement "social distancing" to limit the spread of the virus.

Mellie · Apr 4, 2006

Re: Chikungunya Outbreak Confined To Bagan Panchor Village

From above

A team has been sent to work with the local authorities there to clean up the villages as the area has been found to have very high aedes breeding index (27 per cent) compared to other areas where the index would be normally around one per cent.

AlaskaDenise · Apr 4, 2006

Re: Chikungunya Outbreak Confined To Bagan Panchor Village

CAkidd said:
.........outbreak of rare mosquito-borne disease,....,

has so far only been detected in Bagan Panchor village in Pantai Remis,
Perak and has not spread to any other places in the country.......,

believed to be brought in by foreign workers, working in the area had been implemented.....

So far, 200 villages have been infected with the Chikungunya .........

Am I missing something?

- it's mosquito borne, but it was brought in by foriegn workers.

- it's only detected in Bagan Panchor & has not spread, but 200 villages have been infected.

Wish reporters would check their stories.

.

tsalagiT · Apr 4, 2006

Re: Chikungunya Outbreak Confined To Bagan Panchor Village

CAkidd said:
April 04, 2006 19:47 PM

Chikungunya Outbreak Confined To Bagan Panchor Village

Dr Chua said several measures to contain the outbreak, believed to be brought in by foreign workers, working in the area had been implemented.

hmmmm. DB..you did say this was a strickly mosquito born disease, correct? this sounds like they are referring to this as being human-human transmitted. I saw you waving the red flag about this very thing quite awhile ago. I remained neutral in the debate, but it's getting more difficult to continue to think perhaps you were wrong. Your recent graphs (BF/1918) and corresponding information is alarming. Glad you've a good smigen of tenacity in ya.

DB · Apr 4, 2006

Re: Chikungunya Outbreak Confined To Bagan Panchor Village

tsalagiT said:
hmmmm. DB..you did say this was a strickly mosquito born disease, correct? this sounds like they are referring to this as being human-human transmitted. I saw you waving the red flag about this very thing quite awhile ago. I remained neutral in the debate, but it's getting more difficult to continue to think perhaps you were wrong. Your recent graphs (BF/1918) and corresponding information is alarming. Glad you've a good smigen of tenacity in ya.

tsalagiT,

I said this is NOT a mosquito born disease.

For the record, and am one of the most tenacious people you will ever meet in your life.

tsalagiT · Apr 5, 2006

Re: Chikungunya Outbreak Confined To Bagan Panchor Village

DB
You may have just met someone who can give you a run for your tenacious title crown. Been said I'm worse than a pit bull....just won't let go till i'm ready. It's also gotten me a few jobs I wouldn't have had a chance at otherwise.

OK. i need some clarification. I'm new to this game. Been concentrating solely on BF. What is your opinion? Is this disease actually Chikungunya....which has acquired the ability to transmit human to human.. Hate to say what i've been thinking is a possibility, but here goes. Is it possible that chikungunya has recombined with another virus? therefore gaining the h-h ability. Like ....BF? first mild wave would easily fit this outbreak, hence low lethal rates. maybe if it's recombined with something, it could well be something else altogether, but BF has been reported in the same places as this chikungunya. this has got me worried, and I don't say that easily. Please tell me I've just got a case of being sentinel looking for the first wave and I'm jumping the gun. Not that this disease going pan wouldn't be bad enough.

DB · Apr 5, 2006

Re: Chikungunya Outbreak Confined To Bagan Panchor Village

Let me put it this way.

I have SERIOUS doubts that the disease that is now spreading like wildfire is Chikungunya.

Dont forget:

H5N1 presents like Dengue.
Dengue presents like Chikungunya.

Therefore.....H5N1 presents like Chikungunya.

tsalagiT · Apr 5, 2006

Re: Chikungunya Outbreak Confined To Bagan Panchor Village

Thanks for the input. You've just made my heart stop.

****. (sorry...i even erased what I wrote first.)

DB · Apr 5, 2006

Re: Chikungunya Outbreak Confined To Bagan Panchor Village

I hate delivering bad news to good people.

Maybe that is why I didn't become a doctor.

tsalagiT · Apr 5, 2006

Re: Chikungunya Outbreak Confined To Bagan Panchor Village

perhaps if you'd become a doctor...we wouldn't have had you here watching for us.

don't feel bad about sharing the news. a day not learning something is a day wasted. and i can't think of anything more important to learn about.

wado, friend
~T

Mellie · Apr 5, 2006

Re: Chikungunya Outbreak Confined To Bagan Panchor Village

Seems to me that sequencing the virus would clarify what is what as far as whether or not recombinations have occurred.

As I understand it, the disease is endemic to the areas it is currently being found in, albeit in much higher incidence than usual.

What if the bird flu (going pandemic) cycle is occurring because of human, animal, viral density and environmental conditions on earth and is part of some cycle of the earth re-establishing a carrying capacity balance... If so, it would make sense in the larger scheme of things that there would be other micro organisms, independent of the bird flu virus that might be experiencing the same opportunities and selective pressures as the birdflu viruses are.

This could simply be a kind of convergence, not necessarily a genetic overlapping.

Let me repost Florida1's post on the bio and range of Chik below ... It's from another thread.

DB · Apr 5, 2006

Re: Chikungunya Outbreak Confined To Bagan Panchor Village

Mellie,

These are my scenerios for how chikungunya has become what it has become.

http://www.flutrackers.com/forum/showthread.php?t=3765

Here is a little write up the effect of Bird flu on its envrionment.

http://www.flutrackers.com/forum/showthread.php?t=35

Mellie · Apr 5, 2006

Re: Chikungunya Outbreak Confined To Bagan Panchor Village

Journal of General Virology <hr size="1" style="color: rgb(204, 204, 204);"> Re-emergence of chikungunya and o?nyong-nyong viruses: evidence for distinct geographical lineages and distant evolutionary relationships

Ann M. Powers1, Aaron C. Brault1, Robert B. Tesh1 and Scott C. Weaver1

[SIZE=-1]Department of Pathology and Center for Tropical Diseases, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0609, USA1 [/SIZE]

[SIZE=-1]Author for correspondence: Ann M. Powers. Fax +1 409 747 2415. e-mail ampowers@culex.utmb.edu<script type="text/javascript"></script> [/SIZE]

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Abstract

References
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Chikungunya (CHIK) virus is a member of the genus Alphavirus in the family Togaviridae. Serologically, it is most closely related to o?nyong-nyong (ONN) virus and is a member of the Semliki Forest antigenic complex. CHIK virus is believed to be enzootic throughout much of Africa and historical evidence indicates that it spread to other parts of the world from this origin. Strains from Africa and Asia are reported to differ biologically, indicating that distinct lineages may exist. To examine the relatedness of CHIK and ONN viruses using genetic data, we conducted phylogenetic studies on isolates obtained throughout Africa and Southeast Asia. Analyses revealed that ONN virus is indeed distinct from CHIK viruses, and these viruses probably diverged thousands of years ago. Two distinct CHIK virus lineages were delineated, one containing all isolates from western Africa and the second comprising all southern and East African strains, as well as isolates from Asia. Phylogenetic trees corroborated historical evidence that CHIK virus originated in Africa and subsequently was introduced into Asia. Within the eastern Africa and southern Africa/Asia lineage, Asian strains grouped together in a genotype distinct from the African groups. These different geographical genotypes exhibit differences in their transmission cycles: in Asia, the virus appears to be maintained in an urban cycle with Aedes aegypti mosquito vectors, while CHIK virus transmission in Africa involves a sylvatic cycle, primarily with Ae. furcifer and Ae. africanus mosquitoes. 

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Introduction

References
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Chikungunya (CHIK) virus, a member of the Alphavirus genus in the family Togaviridae, was first isolated from the serum of a febrile human in Tanganyika (Tanzania) in 1953 (Karabatsos, 1985

). Between the 1960s and 1980s, the virus was isolated repeatedly from numerous countries in central and southern Africa as well as in Senegal and Nigeria in western Africa. During this same period, the virus was also identified in many areas of Asia. Since 1953, CHIK virus has caused numerous well-documented outbreaks and epidemics in both Africa and Southeast Asia, involving hundreds of thousands of people (Halstead et al., 1969a

, b

; Rao, 1966

). CHIK virus infection produces an illness in humans that is characterized by fever, headache, nausea, vomiting, myalgia, rash and arthralgia. Because the clinical symptoms of CHIK infection often mimic those of dengue fever and because CHIK virus circulates in regions where dengue virus is endemic, it has been postulated that many cases of dengue virus infection are misdiagnosed and that the incidence of CHIK virus infection is much higher than reported (Carey, 1971

). 
In Africa, CHIK virus appears to be maintained in a sylvatic cycle involving wild primates and forest-dwelling Aedes spp. mosquitoes. Serological studies have repeatedly demonstrated the presence of antibodies in humans and wild primates throughout the moist forests and semi-arid savannas of Africa (Adesina & Odelola, 1991

; Jupp & McIntosh, 1988

; Rodhain et al., 1989

; Salim & Porterfield, 1973

; Karabatsos, 1975

). To date, a vertebrate reservoir or sylvan transmission cycle has not been identified outside Africa, supporting the historical evidence (Carey, 1971 ) that CHIK virus originated in Africa and was subsequently introduced into Asia, where it is now typically associated with Ae. aegypti mosquitoes. Strains from Africa and Asia are reported to differ biologically (Jupp & McIntosh, 1988

), indicating that distinct lineages may exist. 
In 1996, a closely related alphavirus, o?nyong-nyong (ONN) virus, caused a major epidemic in southern Uganda (Lanciotti et al., 1998

). This was the first epidemic of ONN virus infection since 1959, when a large epidemic swept across East Africa involving over 2 million reported cases (Johnson, 1988

). Unlike CHIK and all other alphaviruses, ONN virus is unique in its transmission patterns: the virus is not transmitted by culicine mosquitoes, but rather by anophelines, typically Anopheles funestus and An. gambiae. A vertebrate reservoir for ONN virus has not yet been identified. The transmission of ONN virus by two common vectors that inhabit much of tropical Africa and that live in close association with humans may be a factor in the rapid spread of the virus during epidemics. 
With the exception of information derived from a limited number of serosurveys, little is known about the relationships of CHIK and ONN viruses (Chanas et al., 1979

; Karabatsos, 1975

; Porterfield, 1961

). ONN is considered to be a subtype of CHIK virus: serological tests reveal a one-way antigenic cross-reactivity between the two agents. Antibody to CHIK virus reacts almost equally with both CHIK and ONN viral antigens while ONN virus antibodies react weakly against CHIK virus antigen (Blackburn et al., 1995

; Chanas et al., 1979

; Lee et al., 1997

; Karabatsos, 1985

). It was once postulated that mutations in CHIK virus led to the emergence of ONN virus and its ability to be transmitted by anopheline mosquitoes (Johnson, 1988 ). However, genetic studies by Lanciotti et al. (1998) as well as the phylogenetic analyses presented here clearly demonstrate that ONN and CHIK viruses are genetically distinct. The phylogenetic and serological studies presented here were designed to help elucidate the evolutionary relationships of these viruses and to aid in understanding their epidemic and maintenance transmission patterns. 

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Methods

References
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Virus preparation.
The CHIK and ONN virus strains used in this study are described in Table 1

. Viruses were diluted and grown on either BHK-21 or Vero 76 cells at an m.o.i. less than 1. After approximately 75% of the cells exhibited cytopathic effects, the virus present in the supernatant was concentrated by polyethylene glycol precipitation (Killington et al., 1996

). The virus pellet was resuspended in 150 ?l TEN buffer and 2 ml Trizol LS (Gibco-BRL) was added in preparation for RNA extraction according to the manufacturer?s protocol. 


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</td><td valign="top" align="left">Table 1. Viruses used in phylogenetic analyses

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RNA extraction and RT?PCR.
RNA was extracted from the virus/Trizol suspension according to manufacturer?s protocols as described previously (Cilnis et al., 1996

). cDNA was synthesized from the RNA using a poly(T) oligonucleotide primer (either T19V or T25V-Mlu; 5' TTACGAATTCACGCGT25V 3'). PCR amplification was performed on the first strand cDNA using the poly(T) primer and a forward primer designed to anneal to genome positions 10344 to 10360 (5' TACCCNTTYATGTGGGG 3') of ONN strain SG650, covering the carboxy-terminal portion of the E2 envelope glycoprotein gene. The following parameters were used: 30 cycles of denaturation at 95 ?C for 30 s, primer annealing at 50 ?C for 30 s, and extension at 72 ?C for 3 min. A 10 min final extension was used to ensure complete product synthesis. 
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Sequencing/phylogenetic analyses.
PCR products ranging from 1?2 to 1?7 kb were isolated from 1% agarose gels. The cleaned DNA fragments were cloned into the pCR2.1 TA cloning vector (Invitrogen) and white bacterial colonies screened for plasmids containing inserts of the correct size. Selected clones were sequenced using the plasmid-specific T7 promoter and m13 reverse primers combined with internal, CHIK virus-specific primers (C3205, 5' GCRACAAACCCSGTAAG 3'; C3152, 5' ACTGGCTRAAAGAACGAGG 3'). Sequencing was performed using an Applied Biosystems Prism 377 sequencer and automated DNA sequencing kit. The deduced amino acid sequences were aligned by using the PILEUP program in the Wisconsin Package (Genetics Computer Group) with default parameters, and the nucleotide sequences were aligned manually based on codon homology. Phylogenetic analyses were performed using maximum parsimony, neighbour joining and maximum likelihood programs implemented in the PAUP 4.0 software (Swofford, 1998

). Distance analyses used the Kimura 2-parameter formula to correct for multiple substitutions of the same nucleotides. Unordered and ordered characters (transition/transversion ratio of 4:1; based on previous alphavirus estimates) were used in the parsimony analysis. Alphaviruses in the Venezuelan equine encephalitis, Barmah Forest and eastern equine encephalitis antigenic complexes were used as an outgroup. Bootstrap analysis (Felsenstein, 1985

) was performed with 1000 replicates to determine confidence values on the clades within trees. 
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Estimation of divergence times.
An average divergence rate for CHIK and ONN virus lineages was estimated by identification of sister-sequence pairs that were robust (bootstrap values >>90%), closely related and isolated at least 7 years apart in the same geographical region. The number of differences in synonymous changes depicted in branch lengths separating each sister sequence from the predicted common ancestor?s sequence was divided by the number of years between isolations to yield rates expressed as changes per nucleotide per year, and several estimates were compared to provide an estimated mean and standard deviation. Synonymous nucleotide divergence estimates for pair-wise sequence comparisons were generated using the formula of Li et al. (1985)

to correct for multiple substitutions of the same nucleotides. 
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Production of immune sera.
Syrian golden hamsters and BALB/C mice were used to generate immune sera to three strains of CHIK virus (37997, Ross and 1455/75) and one strain of ONN virus (Igbo Ora, IbH12628). Animals received a single injection of virus (~105 p.f.u./ml), either intraperitoneally (i.p.) alone or subcutaneously with a mixture of virus and an Ae. aegypti mosquito salivary gland suspension to enhance the infection. Approximately 4 weeks post-inoculation, blood was obtained from the rodents from the retroorbital sinus and tested for antibody to CHIK virus by an immunofluorescent antibody assay (IFA) or by neutralization test (NT). Mice that were positive for CHIK virus antibody by IFA were injected i.p. with sarcoma 180 cells to produce hyperimmune ascitic fluid. Abdominal fluid was removed between 1 and 2 weeks after injection of the sarcoma cells and was used in IFA and plaque reduction neutralization tests (PRNT) to determine homologous titres. 
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Titration of neutralizing antibody.
Three of the four viruses (37997, Ross and 1455/75) generated a detectable homologous antibody response as determined by IFA (in mice) or NT (in hamsters). Only two of these, 37997 and Ross, had IFA titres sufficient to perform additional serological analyses. These two viruses were used in 80% PRNTs to determine both the homologous and heterologous neutralizing antibody titres. 

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Results

References
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Phylogenetic analysis
Cloned PCR products ranging from approximately 1200 to 1700 nucleotides of the E1 envelope glycoprotein gene and the entire 3' noncoding region (NCR) were sequenced and aligned using the PILEUP program in the GCG software package. Because alignment of the 3'NCR was poor, only the E1 coding nucleotides were used in the phylogenetic analyses. Both distance matrix programs and maximum parsimony generated trees with the same basic topology, differing only in the arrangement of the CHIK virus isolates from the Asian clade. 
Initial parsimony analyses revealed that several isolates, previously designated as CHIK virus, were genetically quite distinct from the prototype strain and from all other isolates examined. Inclusion of representative members of the Semliki Forest, Venezuelan equine encephalitis, Barmah Forest and eastern equine encephalitis virus antigenic complexes showed that these viruses were actually ONN (strain IPD A234), Semliki Forest (DAK ArB16878) and Sindbis-like (ArMg812 and B448) viruses. Additionally, CHIK virus strains 3412/78 and C-0392/95 were isolated from patients in Thailand suspected of having dengue virus infection, reinforcing the uncertainties of viral diagnosis based upon clinical presentation. 
All of the CHIK and ONN virus isolates examined formed a monophyletic group within the Semliki Forest virus antigenic complex (Fig. 1

), supported by a 100% bootstrap value. The ONN virus isolates formed a robust, distinct clade (100% bootstrap support) apart from all isolates of CHIK virus. ONN and CHIK virus sequences were approximately 28% and 13% divergent at the nucleotide and amino acid levels, respectively, underscoring the distinct nature of the two virus groups. Igbo Ora virus (strain IbH12628) grouped closely with the other strains of ONN, supporting previous reports that this is indeed an antigenic variant of ONN virus (Lanciotti et al., 1998

). 


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</td><td valign="top" align="left">Fig. 1. Phylogenetic analysis of CHIK and ONN viruses generated by performing a PAUP analysis on the 1050 bp partial E1 gene sequence. To correct branch lengths for multiple substitutions, the Neighbor distance program was used to draw the tree utilizing the topology of the PAUP phylogram. Numbers indicate bootstrap values for the groups to the right. Letter A indicates the hypothetical ancestor used to estimate the time of divergence of Asian CHIK isolates from the African progenitor. The bar indicates horizontal distance corresponding to 5% nucleotide sequence divergence.

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All phylogenetic methods divided the CHIK virus isolates into three distinct genotypes, based primarily on geographical origins. One CHIK virus clade consisted of the isolates from Senegal and Nigeria, forming the West Africa genotype (Fig. 1

). These were quite distinct from the remaining CHIK virus isolates having only 78 to 85% nucleotide sequence identity over the fragment analysed. The remaining CHIK virus isolates formed two clades: one contained strains from central and eastern Africa, while the other contained solely Asian isolates. The paraphyletic grouping of the African CHIK viruses supports the historical evidence that the virus was introduced into Asia from Africa. 
Estimated divergence times
An attempt was made to estimate the average rate of evolution of the CHIK and ONN viruses by comparison of sequences of sister taxa from the same geographical areas. Analysis of individual lineages was not possible because too few strains were available. Sister pairs were chosen that had bootstrap values >>90% and were isolated at least 7 years apart. Using these sequences, an estimated rate of evolution was determined to be 6[FONT=ariel,helvetica]x[/FONT]10-4 substitutions per nucleotide per year with a standard deviation of 4[FONT=ariel,helvetica]x[/FONT]10-4. The same estimate was obtained when pair-wise comparisons included distance corrections using the Kimura two-parameter formula or maximum likelihood (see below). The synonymous rate was 5[FONT=ariel,helvetica]x[/FONT]10-4 (standard deviation 3[FONT=ariel,helvetica]x[/FONT]10-4) and the nonsynonymous rate was 6[FONT=ariel,helvetica]x[/FONT]10-5 (standard deviation 5[FONT=ariel,helvetica]x[/FONT]10-5). Although these estimates were based on only six sister-pair sequences and therefore had a high degree of error, they are similar to those previously determined for neotropical alphaviruses (Weaver et al., 1993

, 1997

; Powers et al., 1997

). Using the synonymous rate and the Ks values computed for CHIK strain comparisons (ranging from 0?12 to 0?25 with standard deviations of 0?03), the Asian genotype evolved from a hypothetical African ancestor (node A, Fig. 1

) an estimated 50 to 430 (?1 standard deviation) years ago. Ks values for strains from the West African vs East African/Asian genotypes ranged from 0?75 to 0?86 with standard deviations of 0?10 to 0?12. Using these values, the ancestor of all of the CHIK virus strains is estimated to have emerged between 150 and 1350 years ago. Although the divergence time of ONN virus from CHIK virus could not be estimated reliably due to excessive variance in the Ks values resulting from near saturation of synonymous changes, divergence of CHIK and ONN viruses probably occurred at least thousands of years ago. 
A potential flaw in these time estimates is that substitution rates may vary across nucleotide sites, including synonymous sites, as has been reported for human immunodeficiency virus (Leitner et al., 1997

). Unequal substitution rates across sites could result in an underestimation of true sequence divergence because the sites undergoing more change may accumulate more sequential mutations than are estimated by traditional formulas that assume equal rates. Therefore, we estimated the gamma distribution shape parameter for unequal rates using maximum likelihood analysis applied to all equally parsimonious tree topologies as well as the topology generated by neighbour joining. The gamma distribution shape parameter estimate was 0?42, and the transition/transversion ratio estimate was 4?3, similar to previous alphavirus estimates using substitution data from parsimony analyses (Cilnis et al., 1996

; Weaver et al., 1994

, 1997

). We used these values to generate trees with maximum likelihood branch lengths applied to tree topologies generated using maximum parsimony and neighbour joining methods. Using this approach, similar divergence time estimates were obtained, with the Asian CHIK virus genotype emerging between 50 and 310 years ago, and the West and East African genotypes diverging 100 to 840 years ago. 
Antigenic analysis
To determine the antigenic relatedness of viruses in the CHIK and ONN virus clades, one virus from each CHIK and ONN genotype was selected and used to generate antibodies in hamsters and mice (Table 2

). Four weeks after a single injection of virus, animals were bled, and their sera tested for antibodies by IFA. The three CHIK viruses all produced specific antibodies while the ONN virus-infected mice and hamsters produced no detectable antibody response. The homologous IFA titre of CHIK virus strain 1455/75 was too low to be useful in neutralization assays; however, strains 37997 and Ross produced adequate antibody titres and were tested by 80% PRNT (Table 3

). Results indicated that these viruses have a greater than 4-fold difference in one direction suggesting that they are distinct enough to be classified as antigenic subtypes (Calisher & Karabatsos, 1988

; Calisher et al., 1980

). While no antibody against ONN virus was generated here, eliminating the possibility of performing two-way cross neutralization tests between CHIK and ONN viruses, it would be reasonable to assume that because distinct genotypes of CHIK virus are sufficiently different antigenically to be considered subtypes, the ONN virus lineage would be more likely to be considered a distinct group of viruses within this antigenic complex. 


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</td><td valign="top" align="left">Table 2. Initial serological analysis of CHIK/ONN viruses

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</td><td valign="top" align="left">Table 3. Serological analysis of CHIK viruses (80% PRNT titres)

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Discussion

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Results of our analyses support the hypothesis and historical accounts that CHIK virus probably originated in tropical Africa and subsequently was imported into southern Asia. In Africa, evidence that the virus circulates continually in sylvatic cycles has been documented for decades. The virus has been isolated from sylvatic mosquito species in several countries including Senegal, Cote d?Ivoire, Central African Republic and South Africa (Diallo et al., 1999

; Jupp & McIntosh, 1990

; McCarthy et al., 1996

). The mosquito species involved vary geographically and with ecological conditions. In Senegal, for example, Ae. furcifer, Ae. taylori, Ae. luteocephalus, Ae. africanus and Ae. neoafricanus are the species determined to be of major importance in maintaining CHIK virus transmission cycles. Interestingly, several of these are the same mosquito species involved in maintaining yellow fever virus, perhaps suggesting that outbreaks of CHIK virus infection could be concomitant with sylvan yellow fever (Traore-Lamizana et al., 1996

). In addition, it has been reported that different populations of Ae. aegypti in Senegal have distinct susceptibilities to CHIK virus (Diallo et al., 1999

), suggesting vector strain specificity for CHIK viruses. Further characterization of the mosquito vectors present in the endemic areas and their vector competence for CHIK viruses could provide valuable information regarding the potential for re-emergence of the viruses in human populations. 
In contrast to the numerous species involved in maintenance of CHIK virus infection in Africa, Ae. aegypti and Ae. albopictus are the only vector species known to transmit CHIK virus in Asia. These are urban and peridomestic, anthropophilic mosquitoes that maintain close associations with humans. It is therefore not surprising that outbreaks of CHIK virus infection are noted more frequently in Asia than in Africa. Several studies have demonstrated the varying susceptibility of different Asian mosquito strains for CHIK viruses (Banerjee et al., 1988

; Mourya & Banerjee, 1987

; Mourya et al., 1987

; Tesh et al., 1976

). Because CHIK and dengue viruses are transmitted by the same mosquito species in Asia and because the clinical symptoms of the two viral diseases are similar, the two diseases are difficult to differentiate. Furthermore, there have been documented cases of simultaneous coinfection with CHIK and dengue viruses (Halstead, 1966 ; Myers & Carey, 1967 ), further complicating the characterization of CHIK virus maintenance, evolution and emergence in Asia. 
Another question concerning the transmission of CHIK virus in Asia relates to the high degree of genetic similarity among Asian genotype viruses. Although our sampling of the Asian virus was limited, sequences from viruses spanning a wide geographical range and isolated over a period of almost 35 years showed less than 3% nucleotide sequence divergence (Fig. 1). This genetic conservation in Asia is intriguing for a virus that is known to be maintained only between humans and peridomestic mosquitoes. A similar, high degree of sequence conservation is observed within several other groups of alphaviruses: the North American eastern equine encephalitis viruses (Weaver et al., 1994

; Brault et al., 1999

), Highlands J virus from North America (Cilnis et al., 1996

), western equine encephalitis viruses (Weaver et al., 1997

) and the Sindbis-like viruses distributed throughout Australia (Sammels et al., 1999

). As an example, North American eastern equine encephalitis viruses are maintained by an avian reservoir host; therefore, the increased movement of the virus due to migration of the birds may effectively increase the virus population size and decrease founder effects and genetic drift. This may explain their sequence conservation (Weaver, 1995

; Weaver et al., 1992

; Brault et al., 1999

). It is unknown whether such an avian transmission cycle exists for CHIK viruses in Asia. Migratory patterns of both passerines and shorebirds do encompass much of Southeast Asia ranging from the Yellow Sea and South China Sea across the Philippines and Indonesia to Australia. Additionally, migration routes from India across the Indian Ocean to East Africa have been documented (Williams & Williams, 1990 ). Serological testing of passerines and shorebirds in Southeast Asia could reveal if this is a plausible means of virus dispersal. Alternatively, dispersal of the virus by travel of humans could account for the presence of virtually identical viruses in areas as distant as Indonesia and the Philippines to Barsi in central India, as well as the introduction of the virus into Asia from Africa. 
The phylogenetic results presented here clearly demonstrate that ONN virus did not emerge via a recent mutation of CHIK virus as was once postulated (Johnson, 1988

). This hypothesis was based on serological evidence indicating that the viruses could only be distinguished by two-way specific antigenic tests (i.e. neutralization assay) or the use of monoclonal antibodies (Karabatsos, 1975

; Porterfield, 1961

). Antiserum raised against CHIK virus reacted with ONN virus but the reciprocal was not true, leading to the hypothesis that mutations in CHIK virus generated altered structural configurations in ONN virus affecting seroassay results (Johnson, 1988

; Williams & Woodall, 1961

; Williams et al., 1962

). It was suggested that these same mutations were responsible for the novel ability of ONN virus to replicate in and be transmitted by anopheline mosquitoes. However, if ONN virus undergoes periodic emergence from CHIK virus progenitors, ONN virus isolates from the outbreak in Uganda in 1996 would be predicted to group phylogenetically with CHIK virus isolates rather than with the other strains of ONN virus as seen in our analysis (Fig. 1

). For example, repeated emergence from a common progenitor has been found with epidemic/epizootic Venezuelan equine encephalitis viruses, which emerge periodically from enzootic viruses and occupy clades nested within the enzootic ID lineage (Kinney et al., 1992

; Powers et al., 1997

; Weaver et al., 1996

). 
In addition to the antigenic and sequence differences between CHIK and ONN viruses, differences in several other biological patterns exist. Studies examining the relative ability of various strains of CHIK and ONN to replicate in different cell types have shown clear distinctions between these two viruses. CHIK viruses can replicate in both Ae. aegypti cell lines and numerous Aedes spp. mosquitoes (Chanas et al., 1979

; Jupp & McIntosh, 1988

; Mourya et al., 1987

) while ONN does not appear to replicate in Ae. aegypti cells (Chanas et al., 1979

). Interestingly, both CHIK and ONN viruses can replicate in An. gambiae cells; however, only ONN replicates in and is believed to be transmitted primarily by An. gambiae or An. funestus mosquitoes under natural conditions (Corbet et al., 1961

; Williams et al., 1965

). Differences between the plaque sizes of CHIK and ONN viruses on mammalian cells have also been described (Chanas et al., 1979

; Tesh et al., 1976

); however, among CHIK and ONN viruses, plaque size may be strain specific (Chanas et al., 1979

). 
A putative explanation for the varying biological properties among CHIK and ONN viruses is differences in the 3'NCR. All alphaviruses sequenced have repeat sequence elements in the 3'NCR that vary in length and number, often according to serogroup (Pfeffer et al., 1998

; reviewed in Strauss & Strauss, 1994). Within some virus groups (e.g. the Sindbis-like viruses) very little sequence heterogeneity is detected in the 3'NCR (Shirako et al., 1991

) while other alphaviruses including Ross River virus, Venezuelan equine encephalitis and Semliki Forest complex viruses show high degrees of 3'NCR variation in both length and nucleotide composition (Faragher & Dalgarno, 1986

; Pfeffer et al., 1998

). Kuhn et al. (1991)

have shown that changes in the repeat sequence elements can affect virus replication in different cell types, suggesting that this region may be important in binding cellular proteins utilized during virus replication. Our sequences from 22 strains of CHIK and ONN viruses from a diverse geographical and temporal range demonstrated such a high degree of variability in the 3'NCR that nucleotide sequence alignments in this area were unreliable. This information, combined with the knowledge that the replicative ability of a given CHIK or ONN viral strain varies tremendously with different strains of mosquitoes, may support Kuhn?s hypothesis. 
http://vir.sgmjournals.org/cgi/content/full/81/2/471

Florida1 says: It would be nice if the genetic data from the chik in La Reunion was released so that a comparison could be made to the chik in India.
From this thread: http://www.flutrackers.com/forum/showthread.php?p=4362#post4362

Chikungunya Outbreak Confined To Bagan Panchor Village

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