Tulane Virus as a Human Norovirus Surrogate - Xi Jiang, Ph. D. Cincinnati Children s Hospital Medical Center University of Cincinnati College of ...

Tulane Virus as a Human
  Norovirus Surrogate
               Xi Jiang, Ph. D.

 Cincinnati Children s Hospital Medical Center
  University of Cincinnati College of Medicine
                 Cincinnati, OH

Tulane virus
• Isolated in stools of rhesus macaques captured in Tulane Primate Center
• Proposed to be a new genus Recovirus
• Genetically closest to human NoVs than to other CV genera
• Replicate in monkey kidney cells (LLCMK-2)
• Recognizing human HBGA (B antigen) like human NoVs
• A reverse genetic system has been developed
• Experimental challenge of Rhesus Macaques with TV has resulted in infection
  and illness in 2 of 3 animals (Sestak et al., PLoS One, 2012)
• Cryo-EM structure of the prototype TV has been resolved

• With the funding of this USDA NIFA project, a number of
  important reagents have been generated:
   • Antibodies against individual structural and
      non-structural proteins
   • Monoclonal antibodies against TV
   • Adaptation of TV into several monkey kidney
       cells

                                   Farkas et al., 2008; Wei, et al., 2008; and Yu et al., submitted

Comparison of TV with other HuNoV
        surrogates (MNV and FCV)

• Thermal inactivation
• Stability to extreme pH
• Disinfection, alcohol, bleach, UV
• Long-term persistence in fecal matrix in environmental
  conditions
• Organic solvent stability
• Real-time RT-PCR and plaque assay
• Duplicate samples undergo RNase treatment before RNA
  extraction

Heat treatment
•   Variable temperatures:
     – 37˚C – internal body temperature
     – 56˚C – low temp / long time pasteurization
     – 72˚C – high temp / short time pasteurization

•   37˚C had little impact on MNV infectivity, but decreased TV PFU/mL by > 3
    log after 4 days and no infectious FCV was observed after 48 hours

•   56˚C incubation significantly decreased log PFU/mL after 5 minutes for TV
    and FCV and after 15 minutes for MNV

•   72˚C heat significantly decreased PFU/mL > 3 log after 15 minutes for all
    three viruses

72˚C
1.2                                                            1.2

 1                                                              1

0.8                                                            0.8

0.6                                              TV            0.6                                                 MNV
                                                 TV + RNase                                                        MNV + RNase
0.4                                                            0.4

0.2                                                            0.2

 0                                                              0
      TV    72C, 0.5 min 72C, 1 min 72C, 2 min                          MNV   72C, 0.5 min 72C, 1 min 72C, 2 min
1.2

 1

0.8
                                                                     • 72˚C incubation resulted in little

0.6                                              FCV                    change in overall RNA levels
                                                 FCV + RNase
0.4                                                                  • RNase treatment resulted in greater
0.2
                                                                        decreases in RNA for TV and FCV,
 0
      FCV   72C, 0.5 min 72C, 1 min 72C, 2 min                          indicating capsid damage

Acidic environment
• Since HuNoV and TV must survive the extremely low pH of the
   stomach to infect the intestine, the effect of pH3 on each virus
   was studied
• pH 3 has little effect on overall RNA levels for all three viruses,
   but significantly decreases viral titer (PFU/mL) of FCV
• TV infectivity was reduced at 37˚C for 2 hours, but the level is still
   likely infectious
• Additional time-points and pH levels (especially pH2 and pH 9
   and 10) will be tested

Ethanol
•   40 and 70% EtOH appear to affect RNA levels at long timepoints (30
    min), but little effect was seen at 15 sec
•   RNase treatment showed capsid damage to TV at 30 min for 40 and
    70% EtOH but little at 15 sec
•   TV infectivity was affected by 70 and 90% EtOH at 30 min
•   While 40 and 70% EtOH were effective for MNV, only 70% EtOH for
    FCV
•   TV PFU/mL decreases ~ 2 logs with 70% EtOH and no infectious
    virus was observed with 90% EtOH after 5 minutes
•   70 and 90% EtOH appear to greatly affect TV capsid integrity under
    dried condition

Bleach
•   Various concentrations of bleach at 5 min contact time were tested
     – 10% bleach – 5842 ppm free chlorine
     – 0.5% bleach – 292.125 ppm free chlorine
     – 0.01% bleach – 5.8425 ppm free chlorine
•   For all 3 viruses, only 10% bleach appeared to have a significant effect on
    RNA level
•   RNase treatment did not appear to have an added effect, so when bleach
    affects capsid integrity it also affects the RNA
•   Interestingly, only 10% bleach had a significant impact on TV and FCV,
    while 0.5% bleach had significant effect on MNV (~ 2 log drop)

•   All above experiments will be repeated, including treatment with UV, organic
    solvents and virus survival at different temperatures and in liquid and on dry
    surfaces

Epochal evolution of GII.4
• New variants of GII.4 appeared every 2-4 years, resulting
  in major epidemics
• Selection by the herd immunity, similar to that of influenza
  virus
• Trade-off for new HBGA binding properties
• Strongly selection by HBGAs, while mild antigenic
  vatiation may occur, the major secretor binding
  patterns are not changed
• A critical issue on virus transmission, epidemiology and
  disease control and prevention

Evolution of NoVs selected by HBGAs
• Two levels of divergent
  evolution
   - five genetic lineages
     (genogroups)
   - multiple sublineages
     (genotypes)
• Each lineage and
  sublineage must have
  traveled hundreds and
  thousand years
• Well adapted with
  optimal fitness
• Running out sequence
  spaces
• Not easily jump from
  host to host and from
  one binding pattern to
  others

GII.4 is one of the earliest
sub-lineages after GII NoVs
were introduced to humans

The receptor binding interfaces of GII.4
      NoVs are highly conserved

   • GII.4 underwent only minor antigenic variations, natural flocculation
   • Future studies by continual surveillance are necessary

Acknowledgment
Christina Quigley
Wen Lei
Marissa Choi
Dongsheng Zhang
Weiming Zhong
Jeff Wei
Qiang Fan
Ming Tan

Funding: USDA-NIFA