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Biotech / Medical : SARS and Avian Flu -- Ignore unavailable to you. Want to Upgrade?

To: Biomaven who wrote (1128)	1/2/2004 9:52:54 AM
From: Henry Niman	Respond to of 4232

Here is a bit more analysis on the sequence information released showing that the Guangzhou isolated is about 99% homologous to other SARS CoVs in the S, M, and N genes. The data essentially eliminates the possibility that the sequence is from lab contaminants because contaminants would produce exact matches (there could be a few differences due to sequencing errors, but those difference would be far less than the 1% reported). The reported numbers indicate that there are 22 or 23 differences in the S gene (relative to the closest Genbank match), 6 or 7 differences in the M gene, and 10-11 differences in the N gene (these assume that the reported differences are not due to major insertions or deletions, but those types of changes generally have not been found in these genes). Thus the 99% homology would indicate that the sequence is not a lab contaminant and the virus is in the SARS CoV group of coronaviruses (other animal coronavirus generally have 20-30% sequence homology). However, although the sequence is from a SARS CoV, the number of differences is on the high side, raising the distinct possibility that the virus came from a reservoir other than a masked palm civet. Comparison of the civet sequences to human sequences shows that most of the differences are concentrated in the S gene. The civets have 13 S gene polymorphisms that are found in 100% of the reported human isolates. There are 9 more polymorphism that are changed in almost all human isolates and 13 more found scattered in smaller regionally linked isolates. The new Guangzhou sequences has 22-23 changes relative to other SARS CoVs. If it came via civets, it would have the 13 polymorphisms found in all human isolates, so an additional 22-23 is quite a few. Some of these may be unique (the above changes did not include unique changes - all changes in human SARS CoVs described above were found in 2 or more isolates), so it is possible that the new isolate was linked to the civets, but just had quite a few more changes than other isolates. However, the M and N genes are much more conserved in the SARS CoV sequences at Genbank, and differences in these genes raise the possibility that the recent isolate has a different animal reservoir. In the N gene there are no differences between the civet and human sequences that are widespread. There are 3 differences that are found in a small number of human isolates. The new Guangzhou sequence however appears to have 10-11 changes, which is much higher than any reported human SARS CoV. Thus it is these differences in the N gene that raise the possibility that the new Guangzhou sequence is actually a VERY closely related SARS CoV cousin. One of the key questions about the current case relates to the source of the infection. The index case was reported to have had suspect SARS last March, but was not diagnosed with probable SARS. Thus, the two SARS-like conditions may have been unrelated. However, it seems possible that the SARS CoV partially sequenced was the same virus that caused the earlier symptoms, but the 1% difference made it more difficult to lab confirm the earlier suspect SARS condition. If the virus was not completely eliminated, then the current pneumonia could represent an re-activation of the virus. The full sequence, or even the actual sequence completed thus far, will more precisely define the above possibilities, but at this time it seems clear that the Guangzhou patient was infected with a SARS CoV whose origin is not yet well defined.

To: Biomaven who wrote (1128)	1/2/2004 3:02:56 PM
From: Henry Niman	Respond to of 4232

The media is finally catching up with the fact that the Guangzhou index case is clearly infected with a novel SARS CoV, although the focus below is on the S gene. The % homology reported below indicate that the N gene is also unique, and this may be generating some difference in antibody tests if different labs are using different assays that measure antibodies to the N gene product. The fact that the patient previously had SARS symptoms but was not confirmed to have probable SARS also looms large. This may indicate that a new set of diagnostic reagents will be required to detect this novel SARS CoV, which thus far has not been isolated in culture. The S gene sequence is the same length as SARS CoV sequences at Genbank and the % homology clearly shows that this novel SARS CoV is a VERY close cousin of other sequenced SARS CoV. However, the likelihood that it resides in another animal reservoir remains high and its ability to cause a fatal pneumonia remains to be demonstrated. However, the possibility that it has caused a SARS-like condition in the same individual twice within 9 months is a significant concern. Such a condition may end up being called SCRS (Severe Chronic Respiratory Syndrome). Virus variation found in suspected SARS case ( 2004-01-02 02:07) (Xinhua) The results of a gene sequencing test show the coronavirus found in the suspected severe acute respiratory syndrome (SARS) case in Guangzhou is a virus variation with new sequence, according to the Center for Disease Prevention and Control in southern China's Guangdong Province Friday evening. That's to say, it is possible that the man with the suspected case of SARS in the province have contracted SARS, according to the center. The center had carried out a series of tests on the case. Using the method of reverse transcription-polymerase chain reaction (RT- PCR), scientists obtained S, M and N genes of coronavirus from the suspect. Comparing the sequences of the gene section with all those of the SARS coronavirus published by the international gene bank, experts found that 98.8 to 99.4 percent of 3,768 bases of S gene obtained from the suspect, 99 percent of 658 bases of M gene, and 99 percent of 1,068 bases of N gene, are isogenous with those of S, M and N genes published by the gene bank. "The results point to the possibility of the suspect infected by SARS coronavirus, but our sequence test shows that the S gene is a new sequence that has not been published," said Dr. Chen Qiuxia with the center. Prof. Zhong Nanshan, an academician of the Chinese Academy of Engineering, also said the test result showed a possibility that the SARS coronavirus has a variation. Both Chinese experts and World Health Organization experts have adopted a prudent approach to the final diagnosis of the case while generally agreeing to the results of the tests, Zhong said. "But the wait will continue in the final diagnosis of the suspected SARS case because the infection channel and infectivity remained unknown," he added. www1.chinadaily.com.cn

To: Biomaven who wrote (1128)	1/2/2004 9:23:17 PM
From: Henry Niman	Respond to of 4232

Below is the latest ProMed commentary (the rest of the report was the NY Times story and the report on sequence homologies). Although ProMed is waiting for more laboratory data, it seems that the only informative lab data (other than a complete sequence) would be data on March clinical samples in association with the patient's suspect SARS diagnosis. A SARS diagnosis has been laboratory confirmed many times over now via positive PCR in several labs, as well as SARS CoV antibodies and a rising SARS CoV titer. The only outstanding question mark was the possibility of lab contamination, and now that 3 genes have been sequenced, contamination has been ruled out because the sequences are only 99% homologous to reported SARS CoVs, and the Guangzhou sequence is that of a novel SARS coronavirus that has never been previously reported. Thus, at this point it is clear that the Guangzhou case is the SARS index case for this season. The major outstanding issue, which is the real reason behind the delay in an official confirmation, is the source of the novel SARS CoV that caused the SARS clinical symptoms on Dec 16. Was it acquired recently or is it from an activation of the March infection. ProMED-mail <promed@promedmail.org> [A WHO update on 30 Dec 2003 mentioned that laboratory testing as of that date was inconclusive who.int with the recommendation that further tests are needed. In a discussion on the epidemiologic investigation as of that date, it was stated that "All his relevant human contacts identified so far are well. Some have already been removed from medical observation. Others remain in quarantine, but are expected to be released over the next few days, as the 14-day observation period mandated by China's health authorities draws to a close." We await news of additional laboratory testing. - Mod.MPP]

To: Biomaven who wrote (1128)	1/2/2004 10:09:37 PM
From: Henry Niman	Read Replies (1) \| Respond to of 4232

The news reports on the Guangzhou SARS case are getting more and more entertaining by the hour. The latest report indicates that the Guangzhou SARS index case was "slightly exposed" to the SARS coronavirus at some point. It would be interesting to know how a "slightly exposed" determination was made, when the source of the infection is unknown. I suspect that the "slightly exposed" characterization is somehow related to a low titer, most likely of neutralizing antibodies. However, the level of neutralizing antibodies cannot be measured because the novel SARS CoV has not been isolated. The neutralizing antibodies are directed against the Spike protein and the entire S gene has been sequenced. As noted in several of the media reports, the novel coronavirus is 98.8 to 99.4% homologous to the SARS CoV S gene sequences at Genbank. That means that there are 22-44 nucleotide differences in the S gene when the novel coronavirus is compared to previously reported isolates. Since the index case made neutralizing antibodies to the novel coronavirus, the low titer is not due to a "slight exposure", but instead is due to antigenic drift. This is well illustrated by recent data on antibody titers against the Fujian and Panama influenza viruses. Ferrets were exposed to the Panama strain and generated a robust titer of 1280. However, when that antisera was tested on the Fujian strain, the titer was only 40-80. The low titer was not due to a "slight exposure" of the ferrets to Fujian, but instead was due to the fact that most of the neutralizing antibodies against Panama failed to recognize Fujian. The number of nucleotide differences in the HA gene between Panama and Fujian is 13, so a difference of 22-44 nucleotides between the antibody SARS CoV test isolate and the novel Guangzhou isolate is significant. even after correcting for the fact that the HA of flu is about 1/3 of the Spike protein of SARS CoVs. Thus, it seems that more effort should go into finding the source of the Guangzhou infection and less effort into coming up with inaccurate descriptions of the novel SARS CoV infection. chinapost.com.tw China patient may have been exposed to SARS 2004/1/3 BEIJING, Agencies The World Health Organization (WHO) said Friday initial tests suggested a suspected Chinese SARS patient may have been "slightly exposed" to the SARS coronavirus at some point. This was the preliminary conclusion from a virus neutralization test, which has been used only once so far, the WHO said in a statement, adding a final diagnosis might take days. "The results from that neutralization test indicated that the patient may have been slightly exposed to the SARS coronavirus at some point, but it was impossible to determine exactly when," it said.

To: Biomaven who wrote (1128)	1/6/2004 10:12:04 AM
From: Henry Niman	Read Replies (1) \| Respond to of 4232

The last few weeks has led to an outbreak of CARS (Confused Adult Respiratory Syndrome) over the index case of SARS in Guangzhou for this season, and the reported "full recovery" of the patient, suggests that he may have indeed have had MARS (Mild Adult Respiratory Syndrome) although an earlier respiratory illness in March and lack of an epi-link has led to speculation that he had SCRS (Severe Chronic Respiratory Syndrome), which progressed to MARS. The novel sequence of the SARS CoV has implicated a new lineage, but all of the above, combined with animal sequence data of SARS CoVs and high frequencies of SARS CoV antibodies (in the absence of clinical disease) in market workers, especially those that handle masked palm civets, has led to a culling of civets and related market animals, which has led to concerns over destroying the evidence. I think that the evidence is beginning to paint the following picture: SARS CoVs are novel coronavirus that were not know to the scientific or medical community prior to the SARS outbreak last spring. However, the promiscuous virus has been infecting wild animals for some time and the infections are most prevalent in masked palm civets. The virus can easily pass from the civets to other animals, especially those housed together in open markets, as well as those who sell, slaughter, or cook related animals. This has led to a fairly significant viral gene pool, the mutates frequently. However, prior to last season, these mutations did not produce significant clinical symptoms. Several variants emerged last season. Although the index case developed symptoms in Nov 2002, the epidemic didn't really take off until January 2003. At that time there were several animal to human jumps, leading to clinical manifestations. However, reports indicate that there were only 5 fatalities in the first 305 cases in Guangdong Province. The disease then burst onto the International scene following the transmission at the Metrople Hotel, which was designated as a super spreader event. Additional super spreader event, the Prince of Wales outbreak, led to the Amoy Gardens outbreak and the Flight CA-112 outbreak, which then spread SARS to Taiwan and several cities in Mainland China. Sequencing of the isolated virus indicated that the virus had about 4 dozen mutations relative to the civet sequences and these 4 dozen mutations were found in almost all human sequences, which now exceed 150. However, the current database of sequences was generated using a heavy selection pressure. Most of the isolates grew in culture and caused SARS that was either fatal, highly contagious, or both. Missing from the database would be SARS CoVs causing milder infections (MARS) as well as those that did not grow well in culture. The BJ302 series from Beijing showed that many novel Spike gene mutations could be found with PCR screening. Although these mutations were found in many Beijing patients, they have not been reported in any isolated virus or in any clinical sample from patients outside of Beijing. Thus, the possibility that SARS CoVs can cause a broad range of respiratory illnesses has not been thoroughly investigated. The recent heightened vigilance however, has led to the discovery of a novel SARS case. The novel S gene sequence only has 98.8% to 99.4% homology with previously sequenced SARS CoVs (although this frequency may be higher when compared to animal SARS CoVs) and the virus appears to represent a new animal to human jump. The new jump is consistent with last season's outbreak, which can be seen in the sequences of the three masked palm civet isolates on deposit at GenBank. These three isolates have 19 polymorphisms which are found in all civet sequences but no human sequences, significantly reducing the chance that they are due to a human to civet transmission. However, additional polymorphisms suggest that the civets have some mutations that appear in all or most human isolates, even though the changes are not found in other civets. These civet polymorphisms define some of the key molecular markers. One civet (SZ16) has a marker found just in the raccoon dog sequence (SZ13) suggesting that the racoon dog was infected by the civet. Another civet, SZ1, has a marker that is found in almost all human isolates. It is missing in only the earliest human isolates, suggesting that this marker was introduced at a slightly later date. A third civet (SZ3) has a number of informative markers. It has four changes that are in all human isolates, indicating that these changes happened very early and became extremely well established. It also has two markers found just in one human isolate, GZ43, from an early transmission to a health care worker. SZ3 also has another marker found in CUHK-AG3, a fatal Amoy Gardens case. Media reports suggest that the latest infection in Guangzhou involves yet another lineage which is closely related to yet another animal isolates. A number of additional animal isolates display various degrees of "humanization", similar to the shared markers described above. Thus, the following picture is beginning to emerge. There are many SARS CoVs in wild animals in Guangdong Province. These isolates can produce a broad spectrum of human respiratory diseases. As the virus mutates further, infection and mortality rates can increase. Not all of these animal isolates grow well in culture and not all are easily detected with existing diagnostic probes and these novel isolates may not produce high titers in humans. Using clinical parameters to detect the infections selects for the more mutated versions that produce higher infection rates and mortality. Thus, there may be large numbers of people with SARS CoV infections, who are asymptomatic (such as market workers), have mild respiratory symptoms, have mild SARS symptoms, or have full blown SARS. At this stage of the season, SARS, as seen in February, March, and April of last year, may not have emerged yet. Instead the initial animal jumps are producing a milder disease, not easily detected using classical SARS clinical criteria. There may be many cases of SARS CoV infections in cases with relatively mild conditions, but most of these individuals are not detected because they are not being screened. Thus, at this time of year, SARS CoV infections may be quite prevalent, but full blown SARS is still quite rare. The current screening approaches do not address the spread of SARS CoVs. Instead the focus is on full blown SARS, so much of the SARS CoV evidence in humans is not being identified or analyzed.