J.S.M. Peiris “SARS-COV-1” Paper (2003)

On November 16, 2002, the first case of an atypical pneumonia was reported in Guangdong province in the southern part of China. It wasn’t until nearly 4 months later on March 12th, 2003 that the WHO announced a global alert about a severe pneumonia affecting parts of Asia. On March 24th, 2003, a CDC laboratory analysis suggested that this “new” respiratory disease was caused by a “Coronavirus.” On April 16th, 2003, 5 months after the first reported case of this atypical pneumonia in China, the WHO issued a press release stating that “SARS-COV-1” was the official cause of SARS. Below are highlights from one of the papers cited as evidence for this new “Coronavirus:”

Coronavirus as a possible cause of severe acute respiratory syndrome

“We collected nasopharyngeal aspirates and serum samples from all patients. Paired acute and convalescent sera and faeces were available from some patients. A lung-
biopsy tissue sample from one patient was processed for viral culture and reverse-transcriptase PCR (RT-PCR) and for routine histopathological examination and electron microscopy. We used as controls nasopharyngeal aspirates, and faeces and sera submitted for microbiological investigation of other diseases from patients whose identities were masked.”

“The nasopharyngeal aspirate was assessed by rapid immunoflourescent antigen detection for influenza A and B, parainfluenza types 1, 2, and 3, respiratory syncytial virus and adenovirus, 3 and was cultured for conventional respiratory pathogens on Mardin Darby Canine Kidney, LLC-Mk2, RDE, Hep-2 and MRC-5 cells.4 Subsequently, fetal rhesus kidney (FRhK-4) and A-549 cells were added to the panel of cell lines used. RT-PCR for influenza A5 and human metapneumovirus was done directly on the clinical samples.”

Serology and detection of coronavirus

“After culture and genetic sequencing of a coronavirus from two patients, we developed an RT-PCR to detect the coronavirus sequence from nasopharyngeal aspiration samples. Total RNA from clinical samples was reverse transcribed with random hexamers and cDNA was amplified with primers 5–TACACACCTCAGCGTTG-3-
and 5–CACGAACGTGACGAAT-3- in the presence of 2·5 mmol/L magnesium chloride
(94ºC for 8 min followed by 40 cycles of 94ºC for 1 min, 50ºC for 1 min, and 72ºC for 1 min).

Coronavirus-infected fetal rhesus kidney cells were fixed in acetone and used in an indirect immunofluorescence assay to detect a serological response to the virus.”

Random RT-PCR assay

To find out the genetic sequence information of an unknown RNA virus, we did a random RT-PCR assay. Total RNA from virus-infected and virus-uninfected fetal rhesus kidney cells were isolated. The RNA samples were reverse transcribed with primer 5–GCCGGAGCTCTGCAGAATTCNNNNNN-3-, where N=A, T, C, or G, and cDNA was amplified by a primer 5-GCCGGAGCTCTGCAGAATTC-3-. Unique PCR products (in size) in the infected cell preparation were cloned and sequenced, and the genetic homology compared with those in GenBank.

The routine receipt and inoculation of samples was done in a biosafety level-2 laboratory. Laboratory procedures involving culture of the virus was done in biosafety level-3 containment.”

“Routine microbiological investigation for known viruses and bacteria by culture, antigen detection, and PCR was negative in most cases. Blood culture was positive for
Escherichia coli in one man aged 74 years admitted to intensive care. The finding was attributed to a hospital-acquired urinary-tract infection. Klebsiella pneumoniae and Haemophilus influenzae were isolated from the sputum samples of two other patients on admission.“

“Viruses were isolated on fetal rhesus kidney cells from the lung biopsy and nasopharyngeal aspirate, respectively, of these two patients. The initial cytopathic effect noted was the appearance of rounded refractile cells appearing 2–4 days after inoculation. The cytopathic effect did not progress in the initial culture tubes but on subsequent passage, and appeared in 24 h. The two virus isolates did not react with the routine panel of reagents used to identify virus isolates, including those to influenza A, B, parainfluenza types 1, 2, and 3, adenovirus, and respiratory syncytial virus (DAKO, Glostrup, Denmark). They also did not react in RT-PCR assays for influenza A and human metapneumovirus, or in PCR assays for mycoplasma. The virus was ether sensitive, which shows that it was an
enveloped virus. Electron microscopy of negative stained (3% potassium phospho-tungstate, pH 7·0) ultracentrifuged cell-culture extracts showed the presence of pleomorphic enveloped virus particles of around 80–90 nm (range 70–130 nm) in diameter with surface morphology compatible with a coronavirus (figure 1). Thin-section electron microscopy of infected cells revealed virus particles of 55–90 nm diameter within smooth walled vesicles in the cytoplasm (figure 2, B). Virus particles were also seen at the cell surface. The overall findings were compatible with coronavirus infection in the cells.

A thin-section electron micrograph of the lung biopsy sample from the 53-year-old male contained 60–90 nm viral particles in the cytoplasm of desquamated cells. These viral particles were similar in size and morphology to those observed in the cell cultured virus isolate from both patients (figure 2, A).

The RT-PCR products generated in a random primer RT-PCR assay were analysed, and unique bands found in the virus-infected samples were cloned and sequenced. Of 30 clones examined, one containing 646 bp of unknown origin was identified. Sequence analysis of this DNA fragment suggested this sequence had a weak homology to viruses of the family of Coronaviridae. Deducted aminoacid sequence (215 aminoacids) from this unknown sequence, however, had the highest homology (57%) to the RNA polymerase of bovine coronavirus and murine hepatitis virus, confirming that this virus belongs to the family of Coronaviridae. Phylogenetic analysis of the protein sequences showed that this virus, although most closely related to the group II coronaviruses, was a distinct virus (figure 3).

Based on the 646 bp sequence of the isolate, specific primers for detecting the new virus were designed for RT-PCR detection of this human pneumonia-associated coronavirus genome in clinical samples. Of the 44 nasopharyngeal samples available from the 50 SARS patients, 22 had evidence of human pneumonia-associated coronavirus RNA. Viral RNA was detectable in ten of 18 faecal samples tested. The specificity of the RT-PCR reaction was confirmed by sequencing selected positive RT-PCR-amplified products. None of 40 nasophararyngeal and faecal samples from patients with unrelated diseases were reactive on RT-PCR.”

“If seropositivity to human pneumonia-associated coronavirus in one serum sample or viral RNA detection in the nasopharyngeal aspirates or stools is deemed evidence of infection with the coronavirus, 45 of the 50 patients have evidence of infection. Of the five patients with no virological evidence of coronavirus infection, only one had a serum sample tested more than 14 days after onset of clinical disease.”

Discussion

The outbreak of SARS is unusual in several ways, especially in the appearance of clusters of patients with pneumonia in health-care workers and family contacts.
In this series of patients, investigations for conventional pathogens of atypical pneumonia proved negative. However, a virus belonging to the family Coronaviridae was isolated from the lung biopsy and nasopharyngeal aspirate of two SARS patients and other patients with SARS had a serological response to this virus.”

Phylogenetically, human pneumonia-associated coronavirus was not closely related to any known human or animal coronavirus or torovirus. We based our analysis on a 646 bp fragment of the polymerase gene which showed that the virus belongs to antigenic group 2 of the coronaviruses, along with murine hepatitis virus and bovine coronavirus. However, viruses of the Coronaviridae can undergo heterologous recombination within the virus family and genetic analysis of other parts of the genome needs to be done before the nature of this new virus is more conclusively defined.“

“Most patients who had clinically defined SARS had either serological or RT-PCR evidence of infection by this virus.’

“Five patients who had SARS had no
serological or virological evidence of coronavirus infection. They need to have later convalescent sera tested to define whether they seroconvert subsequently.
However, the concordance of human pneumonia-associated coronavirus with the clinical definition of SARS seems remarkable, given that clinical case definitions are never perfect.

No evidence of human-metapneumovirus infection, by RT-PCR or rising antibody titre, was detected in any of our patients and no other pathogen was consistently detected. It is therefore highly likely that this coronavirus is either the cause of SARS or a necessary prerequisite for disease progression. Whether other microbial or non-microbial cofactors play a part in progression of the disease remains to be investigated.“

“We describe the clinical presentation and complications of SARS. Less than 25% of patients with coronaviral pneumonia had upper-respiratory-tract symptoms.“

“Our clinical description pertains largely to the more severe cases admitted to hospital. We presently have no data on the full clinical spectrum of the emerging
coronavirus infection in the community or among outpatients. The availability of diagnostic tests we describe will help address these questions. In addition, it will allow questions to be addressed about the period of virus shedding (and communicability) during convalescence, the presence of virus in other body fluids
and excreta, and the presence of virus shedding during the incubation period.

The epidemiological data at present seem to suggest that the virus is spread by droplets or by direct and indirect contact, although airborne spread cannot be ruled out. The finding of infectious virus in the respiratory tract supports this contention. Preliminary evidence also suggests that the virus may be shed in the faeces. However, detection of viral RNA does not prove that the virus is viable or transmissible.“

We have provided evidence that a virus in the coronavirus family is the causal agent of SARS. However it remains possible that other viruses act as opportunistic secondary invaders to increase the disease progression, a hypothesis that needs to be investigated further.“

doi: 10.1016/s0140-6736(03)13077-2.

In Summary:

• A lung biopsy was only carried out on one patient
• The researchers used samples from patients with other diseases as a control
• They used numerous cell lines (Mardin Darby Canine Kidney, LLC-Mk2, RDE, Hep-2 and MRC-5 cells as well as fetal rhesus monkey and A-549 cells) to culture respiratory “viruses”
• After cell cultures and genetic sequences from just two patients, they created an RT-PCR test to detect “virus” in others
• Unpurified fetal rhesus Monkeny kidney cell culture was used to determine a serological response
• To determine the genetic sequence for an unknown “virus,” they did a random RT-PCR assay
• There were instances of other “pathogens” found in SARS cases such as E. Coli, Klebsiella Pneumoniae, and Haemophilus Influenza
• “Virus” was “isolated” (i.e. cultured) in fetal rhesus monkey kidney cells from the lung biopsy and nasopharyngeal aspirate from two patients
• CPE did not occur in the first passage but in subsequent ones
• EM images came from the unpurified rhesus monkey kidney cell culture supernatant
• The researchers determined particles in the lung biopsy were similar in size and morphology to those that they found in the culture fluid and decided they must be the same thing
• Of 30 cloned samples from the random primer RT-PCR assay, only one showed a 646 bp fragment of unknown origin
• This sequence had a weak homology to the “Coronaviridae” family
• Deducted aminoacid sequence had the highest homology (only 57%) to “bovine coronavirus” and “murine Hepatitis virus”
• Specific PCR primers were designed based on this 646 bp fragment from one cloned sample from one patient to detect the “virus” in others
• Of the 44 patient samples tested, only 22 were positive for this fragment
• They ponder that if the indirect methods of detecting seropositivity in one serum sample or detecting “viral” RNA in nasopharyngeal aspirates or feces can be used as evidence of infection, only then would 45 of the 50 patients be considered positive for the assumed “virus”
• Five patients had no virological evidence of “coronavirus” infection
• Again, “virus” was only “isolated” (i.e. cultured) from two patients while the rest were deemed positive due to serological tests
• Their analysis was solely based on the unknown 646 bp fragment of one cloned sample (out of 30)
• They claim “viruses” of the “Coronaviridae” can undergo heterologous recombination within the “virus” family and genetic analysis of other parts of the genome needs to be done before the nature of this new “virus” is more conclusively defined
• The researchers state that no other pathogen was consistently detected so it must be this newly discovered “virus” which was never purified/isolated in this study nor proven pathogenic
• However, they state while it is “highly likely” that “SARS-COV-1” is either the cause of “SARS” or a prerequisite for more serious disease, ruling out other microbial or non-microbial co-factors had yet to be investigated
• Less than 25% of patients with “coronaviral” pneumonia had upper-respiratory-tract symptoms (“coronaviruses” supposedly cause upper respiratory disease…but they caveat this by saying these “viruses” can sometimes cause lower respiratory disease so that they can “have their cake and eat it too”)
• The researchers admit that presently they have no data on the full clinical spectrum of the emerging “coronavirus” infection in the community or among outpatients
• They believe diagnostic tests will allow for answers regarding:
  1. The period of “virus” shedding (and communicability) during convalescence
  2. The presence of “virus” in other body fluids and excreta
  3. The presence of “virus” shedding during the incubation period
• They admit that finding “viral” RNA does not prove that the “virus” is viable or transmissible
• They end by stating a “Coronavirus” is the causal agent of SARS yet there could be other “viruses” acting as secondary invaders

“SARS-COV-1” was claimed the casual agent of “SARS” based on cell-cultured evidence from only two patients. Of 30 cloned RT-PCR samples from one of the patients, only one 646 bp fragment of unknown origin showing weak homology to “Coronaviruses” was found and this was subsequently used to create RT-PCR assays for diagnosis. Only 22 out of 50 “SARS” patients tested positive for this 646 bp fragment. Five patients had no virological evidence of “SARS” whatsoever yet were still considered “SARS” cases. Other pathogens were found in some of the patients but these were relegated to possible secondary invaders.

In other words, the same small sample size, the same unpurified cell culture soup, the same unpurified EM images, the same useless serological tests, and the same assumptions/conclusions as the early “Coronavirus” papers but this time with the further indirect PCR and genomic data added in to keep things fresh.

Sadly, still no properly purified/isolated “virus” coming directly from the samples of sick patients, still no proven pathogenicity, and still no proper controls.