Inactivation of highly transmissible livestock and avian viruses including influenza A and Newcastle disease virus for molecular diagnostics

Abstract

There is a critical need for an inactivation method that completely inactivates pathogens at the time of sample collection while maintaining the nucleic acid quality required for diagnostic PCR testing. This inactivation method is required to alleviate concerns about transmission potential, minimize shipping complications and cost, and enable testing in lower containment laboratories, thereby enhancing disease diagnostics through improved turn-around time. This study evaluated a panel of 10 surrogate viruses that represent highly pathogenic animal diseases. These results showed that a commercial PrimeStore® molecular transport media (PSMTM) completely inactivated all viruses tested by >99.99%, as determined by infectivity and serial passage assays. However, the detection of viral nucleic acid by qRT-PCR was comparable in PSMTM and control-treated conditions. These results were consistent when viruses were evaluated in the presence of biological material such as sera and cloacal swabs to mimic diagnostic sample conditions for non-avian and avian viruses, respectively. The results of this study may be utilized by diagnostic testing laboratories for highly pathogenic agents affecting animal and human populations. These results may be used to revise guidance for select agent diagnostic testing and the shipment of infectious substances.

Keywords: animal virus; inactivation; influenza virus; molecular diagnostics; molecular transport media.

Figure 1

Diverse viruses are stable at ambient temperature. Virus recovery over time deposited on a plastic surface at ambient temperature for (A) low pathogenic avian influenza virus (LPAI) H7N3; (B) LPAI H6N1; (C) LPAI H5N3; (D) swine influenza virus (SIV) H3N2; (E) Newcastle disease virus (NDV); (F) vaccinia; (G) swinepox; (H) eastern equine encephalitis virus (EEEV)/Sindbis chimera; (I) bovine viral diarrhea virus (BVDV); and (J) Senecavirus A. Virus recovery was assessed by TCID₅₀ titer using the cell line described in Table 1. Each timepoint was compared to timepoint 0. Significance was determined using Student’s t-test. ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001; ^**** p < 0.0001; ns, not significant. Error bars represent the standard error of the mean (SEM) of triplicate experiments.

Figure 2

PSMTM effectively inactivates diverse animal viruses while maintaining the detection of nucleic acid. (A) Virus recovery after PrimeStore® molecular transport media (PSMTM) inactivation or PBS no-inactivation control. Virus recovery was assessed by TCID₅₀ titer using the cell line described in Table 1. (B) Nucleic acid cycle threshold (CT) detection after PSMTM inactivation or PBS no-inactivation control. (C) Serial passage endpoint nucleic acid cycle threshold (CT) detection after PSMTM inactivation or PBS no-inactivation. The serial passage was completed using the cell line in Table 1 and the description outlined in Methods. Nucleic acid was detected according to the reference in Table 1 and the description in the Methods section. PSMTM-treated viruses were compared to the corresponding no-PSMTM control, and inactivation was assessed using the manufacturer’s recommended conditions. Significance was determined using Student’s t-test. ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001; ^**** p < 0.0001; ns, not significant. Error bars represent the standard error of the mean (SEM) of triplicate experiments. Low pathogenic avian influenza virus (LPAI); swine influenza virus (SIV); Newcastle disease virus (NDV); eastern equine encephalitis virus (EEEV); and bovine viral diarrhea virus (BVDV).

Figure 3

Presence of biological material does not affect the effectiveness of PSMTM on the inactivation or nucleic acid detection of diverse viruses. Viruses were combined with species-appropriate sera or cloacal swabs as described in the Methods section. (A) Virus recovery after PrimeStore® molecular transport media (PSMTM) inactivation or PBS no-inactivation control. Virus recovery was assessed by TCID₅₀ titer using the cell line described in Table 1. (B) Nucleic acid cycle threshold (CT) detection after PSMTM inactivation or PBS no-inactivation control. (C) Serial passage endpoint nucleic acid cycle threshold (CT) detection after PSMTM inactivation or PBS no-inactivation. Serial passage was completed using the cell line in Table 1 and the description outlined in the Methods section. Nucleic acid was detected according to the reference in Table 1 and the description in Methods. PSMTM-treated viruses were compared to the corresponding no-PSMTM control, and inactivation was assessed using the manufacturer’s recommended conditions. Significance was determined using Student’s t-test. ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001; ^**** p < 0.0001; ns, not significant. Error bars represent the standard error of the mean (SEM) of triplicate experiments. Low pathogenic avian influenza virus (LPAI); swine influenza virus (SIV); Newcastle disease virus (NDV); eastern equine encephalitis virus (EEEV); and bovine viral diarrhea virus (BVDV).

Figure 4

PSMTM to virus ratio determines the effectiveness of inactivation. Virus recovery and nucleic acid detection were determined after PrimeStore® molecular transport media (PSMTM) inactivation or PBS no-inactivation control utilizing various treatment to virus ratio conditions: (A) 1:1 ratio; (B) 1:3 ratio; (C) 1:10 ratio; and (D) 1:100 ratio. Virus recovery was assessed by TCID₅₀ titer using the cell line described in Table 1. Nucleic acid was detected according to the reference in Table 1 and the description in the Methods section. PSMTM-treated viruses were compared to the corresponding no-PSMTM control. Significance was determined using Student’s t-test. ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001; ^**** p < 0.0001; ns, not significant. Error bars represent the standard error of the mean (SEM) of triplicate experiments. Eastern equine encephalitis virus (EEEV).

Figure 5

Virus inactivation and nucleic acid detection are stable over time in PSMTM. Virus recovery and nucleic acid detection over 21 days for viruses deposited in a plastic screw-cap tube at ambient temperature. Inactivation was assessed using the manufacturer’s recommended conditions. (A) Virus recovery was assessed by TCID₅₀ for PBS no-inactivation control-treated LPAI H7N3 (left) and PrimeStore® molecular transport media (PSMTM)-treated LPAI H7N3 (right). (B) Virus nucleic acid cycle threshold (CT) detection for PBS no-inactivation control and PSMTM-treated LPAI H7N3. (C) Virus recovery was assessed by TCID₅₀ for PBS no-inactivation control-treated vaccinia virus (left) and PSMTM-treated vaccinia virus (right). (D) Virus nucleic acid cycle threshold (CT) detection for PBS no-inactivation control and PSMTM-treated vaccinia virus. (A,C) TCID₅₀ titer was determined in the appropriate cell line as identified in Table 1. Each timepoint was compared to timepoint 0 for each respective treatment. (B,D) Nucleic acid was detected according to the reference in Table 1 and the description in Methods. PSMTM-treated viruses were compared to the corresponding no-PSMTM control. Significance was determined using Student’s t-test. ^* p < 0.05; ^** p < 0.01; ns, not significant. Error bars represent the standard error of the mean (SEM) of triplicate experiments. Low pathogenic avian influenza virus (LPAI).