The 2009 pandemic A (H1N1) influenza virus was first identified in Mexico in April 2009 and spread world wide over a short period of time. Well validated diagnostic methods that are rapid and sensitive for detection and tracking of this virus are urgently needed. In this study, time course kinetic characterizations of the abundances of all ten genes of the 2009 pandemic A (H1N1) influenza virus in standard virions and infected MDCK cells were monitored. Results showed that the amounts of each gene in infected cells were significantly higher than those in virions, so that cell lysates were more recommended to be the nucleotide materials detection object than virions. Meanwhile, all genes were present in virions in approximately equimolar amounts, whereas the copy numbers of each gene in cell lysates were distinguishing. The abundances of M1 and NP genes were highest and may be the optimized choice for nucleotide detection in infected cells. Furthermore, the most sensitive time point for viral nucleotide detection in cells was 48 to 56 hour post infection. In infected MDCK cells, the total RNAs amounts of NP and NS1 genes began to rise at 3 hours post infection, whereas other eight genes escalated from 8 hours post infection just as the situation of all genes in standard virions. All these data may be useful for more sensitive diagnosis and surveillance of the novel A (H1N1) virus, and might further limit the transmission of this pandemic disease in the future.
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Key words: the 2009 pandemic (H1N1) influenza virus; real-time PCR; virions; MDCK cells; abundance; sensitivity
On June 11, 2009, the World Health Organization raised the global pandemic alert level to phase 6, the pandemic phase, in response to the emergence and global spread of a novel influenza A (H1N1) virus, which emerged in Mexico in early 2009 (Garten, Davis et al. 2009). The transmissibility of this virus was estimated to be higher than that of seasonal influenza viruses (Fraser, Donnelly et al. 2009). The 2009 pandemic A(H1N1) influenza viruses infections have been primarily seen among young and previously healthy adults, which suggesting that they are most vulnerable to infection. To limit community or hospital transmission, as well as to initiate antiviral therapy in time as recommended by the WHO, accurate and rapid diagnosis for confirming infection with the novel A (H1N1) virus is urgently needed and critical.
Polymerase chain reaction (PCR), especially real-time PCR, remains the best choice for early clinical diagnosis method of this virus (2009; Bolotin, Robertson et al. 2009; Carr, Gunson et al. 2009; Chan, Lai et al. 2009; Ellis, Iturriza et al. 2009; Gunson, Maclean et al. 2009; Jiang, Kang et al. 2009; Liu, Hou et al. 2009; Pabbaraju, Wong et al. 2009; Panning, Eickmann et al. 2009; Poon, Chan et al. 2009; Wang, Sheng et al. 2009; Whiley, Bialasiewicz et al. 2009; WHO 2009; Wu, Kang et al. 2009). To date, most research and commercial detection kits for PCR diagnosis of the pandemic A (H1N1) influenza virus are targeted on hemagglutinin (HA) gene to distinguish the novel virus from other subtype influenza viruses (Jiang, Kang et al. 2009; Liu, Hou et al. 2009; Panning, Eickmann et al. 2009; Poon, Chan et al. 2009; Wang, Sheng et al. 2009). Meanwhile, there are also large parts of PCR assays in use target on matrix (M) gene for its high conservation between various strains (Carr, Gunson et al. 2009; Chan, Lai et al. 2009). However, the efficiency and sensitivity of detection based on other genes of this novel virus have never been reported and compared. In the present study, we compared the abundance of all ten genes of the pandemic A (H1N1) influenza virus in supernatant virions and infected MDCK cell lysates by using Real-time quantitative PCR based on SYBR Green dye. Twenty pairs of primers with amplified products were overlapped and covered full length genome were designed. Meanwhile, cell supernatants and lysates were collected continuously from 0 hour to 96 hours post infection (h.p.i) to monitor the time course and kinetic characterization of viral nucleotide material amounts. We hope the results may close two gaps in knowledge about the detection for the pandemic A (H1N1) influenza virus in laboratory: (1) Which time point post infection is most sensitive for virus detection? (2) Which gene possesses the highest abundance in virions and infected cells? New insights into these two problems may lead to new strategies for inhibiting future extensive transmission of the the pandemic A (H1N1) influenza viruses.
Materials and methods
Viruses and cells
Always on Time
Marked to Standard
The 2009 pandemic A (H1N1) influenza virus strain A/California/07/2009 was used in this study. Virus was cultured in Mardin Darby Canine Kidney (MDCK) cells and aliquots were frozen at -80°C. The 50% tissue culture infectious dose (TCID50) was determined by serial titration of viruses in MDCK cells respectively, and the titers were calculated according to the Reed-Muench method (Reed and Muench 1938). All experiments involving the 2009 pandemic A (H1N1) influenza viruses were conducted under biosafety level 3 (BSL-3) conditions, in associated with guidelines of the World Health Organization (http://www.who.int/csr/resourse/publications/swineflu/Laboratorybioriskmanagement.pdf).
Preparation of total RNAs from infected cells and supernatants at each time point
102 TCID50 viruses were added to MDCK monolayers in 35-mm dishes (Corning). After a 60 min adsorption at 37°C, cells were fed with 3ml serum-free minimum essential medium containing tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK) -treated trypsin (0.5Î¼g/ml) (Sigma) and antibiotics (Sigma). This was designated as 0 h.p.i. Other dishes were then incubated at 37°C. At each time point post infection, 100 Î¼l viral supernatants were harvested and clarified from cell debris by centrifugation at 3,000 g for 10 min. Cells were also harvested, and washed twice with PBS, followed by resuspending in 100 Î¼l PBS. Total RNAs in virion and cell lysates at each time point were extracted by using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. RNAs were dissolved in 30 Î¼l diethyl pyrocarbonate-treated water and stored at -80°C. First-strand cDNA was produced by using random primers with 8 Î¼l RNA in a 20-Î¼l reaction mixture containing 200 U Superscript â…¢ reverse transcriptase (Invitrogen).
Standard sample preparation
Eight full-length segments of the 2009 pandemic A (H1N1) influenza virus (PB2, PB1, PA, HA, NP, NA, M2&M1, NEP&NS1) were amplified from A/California/07/2009 strain, and primers were designed based on the genomic sequences of this strain too, with Genbank accession number FJ966976 (PB2), FJ966978 (PB1), FJ966977 (PA), FJ966974 (HA), GQ338390 (NP), GQ377078 (NA) , FJ966975 (M2&M1), FJ969538 (NEP&NS1) respectively (Table 1). PCR products were cloned into the pGEM-T easy vector (Promega) seperately and positive clones were selected. The standard DNAs were extracted and purified by QIAprep Spin Miniprep Kit (Qiagen) and the concentrations were determined with a Lambda 25 UV spectrometer and converted to copy numbers. Each DNA was serially diluted 10-fold from 1010 copies to 101 copies per microliter and stored at -20â„ƒ.
Real-time PCR assays
The Real-time quantitative PCR assays based on SYBR Green dye were performed on StepOne PCR system (ABI) with 2 Î¼l cDNA in a 20 Î¼l reaction mixture which also containing 10 Î¼l of 2Ã- SYBR Green PCR Master Mix (ABI), 1 Î¼l each of 10 Î¼M forward and reverse primers (Table 1), and 6 Î¼l nuclease-free water. Thermal cycling was done under the following conditions: 94°C for 3 min, followed by 35 cycles of 94°C for 30 s, 51-59 °C for 30 s, and 72°C for 45 s. Fluorescence measurements were taken after each cycle. For M2, M1, NEP, and NS1, the PCR products were the full length genes, and for other six genes, there were two to three pairs of primers each which amplified products were overlapped with each other and covered the full length genes respectively (Table 1).
One-way ANOVA with DUNCAN and LSD methods were used for comparing mean viral load by using different primers at different time points (SPSS 11.5 for Windows).
Generation of standard curves for each pair of primers
Each standard DNA was serially diluted 10-fold from 1010 copies to 101 copies per microlitre. Standard curves showed that there were strong linear relationships (r2>0.99) between the logarithms of the copy numbers and the mean CT values of each real-time PCR assay. (Data not shown).
Time course of viral RNAs amounts in virion
To determine the time course of viral RNAs amounts in virion, MDCK cells were infected with the 2009 pandemic A (H1N1) influenza virus strain A/California/07/2009, and 100 Î¼l viral supernatants were harvested and clarified at 0, 0.5, 1, 1,5, 2, 2.5, 3, 4, 6, 8, 12, 24, 32, 48, 56, 72, 96 h.p.i separately. RNAs were extracted and reverse transcribed to cDNAs which were quantified by real-time PCR with twenty primer pairs based on ten viral genes with SYBR Green dye. Quantification results showed that for PB2, PB1, PA, HA, NP, and NA genes, which used two or three primer pairs for quantification, the results of each gene were not totally identical when using diverse primers (Fig. 1a~f), however, statistical analysis by SPSS software indicated that there were no difference among the quantification results of each gene by different primers (P>0.05). The discrepancy in large probability was caused by the different match abilities of each primer set in annealing with the templates. For all ten genes, the total RNAs presented in standard virions in approximately equimolar amounts at each time point (Fig.1a~h), since statistical analysis indicated that there were no difference among them (P>0.05). Time course kinetic curve of the RNAs amounts of each gene in virions began to rise at 8 h.p.i, and quantification results by using PB2-1, PB2-2, PB1-3, NP2, NA1, NA2, M1, M2, and NS1 primers reached their peak copy numbers at 48 h.p.i, whereas PB2-3, PB1-1, PB1-2, PA-1, PA-2, PA-3, HA-1, HA-2, HA-3, NP-1, and NEP primers detected the climax copies at 56 h.p.i. However, the differences of amounts between these two time points were extremely indistinctive (Fig. 1), which suggested that when clinical suspected positive specimens were cultured in MDCK cells, the most sensitive time course for nucleotide materials detection from the viral particles was 48 to 56 h.p.i.
Time course of viral total RNAs amounts in infected cells
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Similarly, infected MDCK cell lysates were harvested, washed twice, and resuspended in 100 Î¼l PBS at 0, 0.5, 1, 1,5, 2, 2.5, 3, 4, 6, 8, 12, 24, 32, 48, 56, 72, 96 h.p.i separately. Real-time PCR results showed that although there were no differences among the quantification results of PB2, PB1, PA, HA, NP, and NA genes respectively by two or three primer pairs each (P>0.05), the abundance of the genome in cell lysates were distinguishing (Fig. 2). Statistical analysis indicated that the amounts of ten genes in cell lysates could be divided into three grades. The first grade included M1 and NP genes. The highest copies at all time points originated from M1 gene, followed by NP gene, with lower abundance than M1 but higher than other eight genes. Amounts of PB2, PB1, PA, HA, NA, M2, and NS1 genes in cell lysates could be classified into the second grade, and there were no significant differences among them (P>0.05). The abundance of NEP gene was the last grade, which was significant lower than other nine genes in infected cells (P<0.01). Meanwhile, time course kinetic curves of all ten genes in infected cells were not uniform either. The total RNAs amounts of NP and NS1 genes were observed to begin to rise at 3 h.p.i, and were significantly predated compared with other eight genes which escalated from 8 h.p.i (Fig. 1 and 2). Quantification results by using PB2-1, PB2-2, PB2-3, PB1-1, PB1-2, PB1-3, PA3, HA1, NP1, NP2, NA1, NA2, M1, M2, and NS1 primers reached their peak copy numbers at 48 h.p.i, whereas PA-1, PA-2, PA-3, HA-2, HA-3, and NEP primers detected the climax copies at 56 h.p.i (Fig. 1). Similar to the situation in virions, the differences of copy numbers between these two time points were extremely indistinctive too, which illustrated that the most sensitive time course for viral nucleotide materials detection in infected MDCK cells was also 48 to 56 h.p.i.
In the 2009 evolving influenza pandemic, rapid and reliable diagnostic methods remains crucial to limit extensive transmission and to initiate therapy. The WHO defines a probable clinical case as one that is confirmed by (1) specific real-time PCR based detection methods, (2) isolation of the pandemic A (H1N1) influenza virus, or (3) detection of 4-fold rise of neutralization antibodies to this virus (WHO 2009). BSL-2 laboratories with BSL-3 practices are recommended for virus isolation and serology, whereas PCR detection requires only a BSL-2 environment. Of the above diagnostic tests, only real-time PCR based detection methods allow rapid detection of this virus within a few hours.
The genome of the 2009 pandemic A (H1N1) influenza virus comprises eight RNA segments of negative polarity (Smith and Hay 1982). Viral mRNAs from segments 1 to 6 are monocistronic and encode proteins PB2, PB1, PA, HA, NP, and NA separately, whereas viral mRNAs derived from segments 7 and 8 are bicistronic and could undergo alternative splicing for protein expressions, and each encodes two proteins, M2 and M1, NEP and NS1, respectively. In the infected cells, virion RNAs (vRNAs) are transcribed into two different types of transcripts (Hay, Lomniczi et al. 1977). The predominant transcripts are the viral messenger RNAs (mRNAs), which contain a 5' cap structure and a 3' poly (A) tail. The viral mRNAs are incomplete copies of the vRNAs in that they lack a copy of the last 17-22 nucleotides at the 5' end of the vRNAs. Another type of viral transcripts, which are complete copies of the vRNAs, are the templates for progeny vRNA synthesis and represent an intermediate in the replication of the viral genome (complementary RNAs, cRNAs). This may explain why the amounts of total RNAs in infected cells were significantly higher than those in virions, and suggested that after clinical suspected positive specimens were cultured in cells, cell lysates were more recommended to be the nucleotide materials detection object than virions in supernatants.
Our results showed that all eight virion RNA segments were presented in standard virions in approximately equimolar amounts, which demonstrated that this influenza virion packaging is an ordered and selective process that each viral particle must and only have one copy of each eight RNA segments to be incorporated. This phenomenon coincided with other influenza viruses, and also suggested that all primers used in this study exhibited equivalent amplification efficiency, which supplied a firm foundation for further investigation in cell lysates. However, the copy numbers of the genome in cell lysates were distinguishing. The highest amounts at all time points post infection originated from M1 gene, and that is the reason why the conventional PCR detection targeted on M1 gene have been widely used for the first-line screening, since they could identify this virus with high sensitivity. In addition, high conservation between various strains was another reason for the popular applying of M1 gene based PCR detection. Our results also showed that apart from M1 gene, NP was an alternative choice as the target for nucleotide materials detection in infected cells, by the reason that its abundance was a minor lower than M1 gene, but much higher than other genes of influenza virus. High conservation also was the merit of the NP gene targeted detection method to be widely used. However, assays targeted on conserved genes such as M or NP were useful to screen patient specimens for influenza, but could not easily differentiate the 2009 pandemic A (H1N1) virus with other subtype influenza viruses. Assays targeted on surface genes, such as HA, still are extremely critical for sub-typing and antigenic characterization.
Another phenomenon in the time course kinetic curve of each gene in virions and infected cells is that the most sensitive time point for viral nucleotide materials detection was 48 to 56 h.p.i. In virions, RNAs amounts of each gene simultaneously began to rise at 8 h.p.i. However, in infected cells, the total RNAs amounts of NP and NS1 genes were observed to begin to rise at 3 h.p.i, whereas other eight genes escalated from 8 h.p.i just as the situation in the virions. We supposed this may be the outcome of the transcriptional characterization of the influenza virus, with which the transcription stage could be divided into different phases. Immediately after infection, primary transcription occurs (Hay, Lomniczi et al. 1977). In this phase, all eight mRNAs are synthesized in equivalent amounts, and this may be 0 to 2.5 h.p.i which reflected in our results. The following is the second transcription phase, which can be further subdivided into early and late phases. In the early phase of the secondary transcription, NS1 and NP RNAs are preferentially synthesized (Hay, Lomniczi et al. 1977; Smith and Hay 1982; Shapiro, Gurney et al. 1987). The reason, however, for the preferential early expression of the NS1 and NP proteins is still unknown. It is possible that NP is required for the replication and transcription of viral RNA. NS1 might be required for the regulation of cellular gene expression. During the late phase, RNAs of each gene are synthesized in equivalent amounts, as required for progeny virus genome, and this may be 8 hours afterwards post infection demonstrated by our data.
In conclusion, time course kinetic characterizations of copy numbers of the novel A (H1N1) influenza viral genome in standard virions and infected MDCK cells were monitored. The data suggested that cell lysates were more recommended to be the nucleotide materials detection object than virions. Meanwhile, the amounts of the genome in cell lysates were distinguishing. M1 and NP genes might be the optimized choice for viral nucleotide detection in infected cells. Furthermore, the most sensitive time point for nucleotide materials detection in cells was 48 to 56 h.p.i. All these results may provide useful data for rapid diagnosis in the early phase of disease course, and to initiate proper therapy for patients, and further controlling of the pandemic disease.
This work was supported by the IndustryÂ FoundationÂ ofÂ MinistryÂ ofÂ Health (200802036) and the National Science and Technology Major Projects of Infectious DiseaseÂ (2009ZX10004-402ï¼Œ2009ZX10004-016).