Thermostability and Immunogenicity of Genotype II Avian Orthoavulavirus (AOaV-1) Isolates from Duck (Anas platyrhynchos) and Parrot (Eclectusroratus)

Round 1

Reviewer 1 Report

The manuscript reports the results of the biological and molecular characterization of two orthoavulavirus isolates. The authors used routine methods and the results seem sound.

Specific comments

Line 200: It is basic, but please specify that these were antibody titers in the blood

Line 206: Please be explicit that time and treatment were the two factors of the two way ANOVA.

Line 281: These are the values of the thermostability testing at 40C.

I would suggest adding Table S1A to the manuscript instead of Figure 6.

Author Response

Thank you for your comments and advice. We have addressed the concerns and made the changes accordingly.

Specific comments

Line 200: It is basic, but please specify that these were antibody titers in the blood

We have made the change. The new sentence read as follows - 

HI titres in blood of experimental chicks were determined before immunization and at 7, 14, 21 and 28 days post immunization (dpi).

Line 206: Please be explicit that time and treatment were the two factors of the two way ANOVA.

The new sentence read as follows - 

Two way ANOVA with interaction was also done along with critical difference (CD) test for pair-wise comparison of mean serum antibody titre in chicks keeping time and treatment as factors.

Line 281: These are the values of the thermostability testing at 40C.

We appreciate the reviewer's insightful suggestion. Line 281 depicts the values of the thermostability testing at 56⁰ C.

I would suggest adding Table S1A to the manuscript instead of Figure 6.

We have made the change.

Reviewer 2 Report

The manuscript of Sangeeta Das et al. describes they have obtained an isolate that is as immunogenic as the current vaccine strain, LaSota, has low virulence to chickens even after adaptation by 6 passages, and is more thermostable than LaSota strain.  Further studies are needed in aspects of propagation and safety as live vaccine before practical application, but this is an interesting discovery as a new candidate for a live vaccine strain that does not require cold chain.  

I would like to confirm a few things.

First, what was used as the HA antigen in the HI test for immunogenicity. To evaluate immunogenicity between virus strains, it is necessary to use the same antigen, for example, a standard strain.   

Then, what is the virus titer used in the safety testing on chickens in this study? What is the specific viral titer, which is ten times higher than the single dose each of strains (line294)?

Table 2 is somewhat difficult to read because there is no separation of columns. Why does rate constant (K) appear twice in the table? How was rate constant (K) obtained. Why is the “initial titer” of experiment of 56°C different from that of 40°C experiment and not consistent with the text (lines 280-281).

Throughout the manuscript, inferior letter should be used for number of EID50 and Log2.

 In line 362, Li et al [40] reported~.

Author Response

Thank you for your comments and advice. We have addressed all the concerns and made the changes accordingly.

I would like to confirm a few things.

First, what was used as the HA antigen in the HI test for immunogenicity. To evaluate immunogenicity between virus strains, it is necessary to use the same antigen, for example, a standard strain.

LaSota vaccine strain is a standard Newcastle disease virus (NDV) strain and it was used as the HA antigen in the HI test for evaluation of immunogenicity. 

Then, what is the virus titer used in the safety testing on chickens in this study? What is the specific viral titer, which is ten times higher than the single dose each of strains (line294)?

In the present study, the chicks  were immunized against chicken adapted PSD44C and PSP91C and commercially available NDV LaSota vaccine at 7 days of age via intra-ocular route with standard dose of 10⁶ EID50 per chick and for the safety test,  ten times the single dose (10⁶ EID50) each of strains were used.

Table 2 is somewhat difficult to read because there is no separation of columns. Why does rate constant (K) appear twice in the table? How was rate constant (K) obtained. Why is the “initial titer” of experiment of 56°C different from that of 40°C experiment and not consistent with the text (lines 280-281).

We apologize for our error. We have changed [The initial HA titre (Log2/50µl) and infectivity (Log10EID50) of PSD44C, PSP91C and LaSota were (7.636, 4.572), (7.274, 4.175) and (8.125, 4.801) respectively.] to [The initial HA titre (Log2/50µl) and infectivity (Log10EID50) of PSD44C, PSP91C and LaSota were (7.869, 4.902), (7.402, 4.334) and (8.582, 4.860) respectively.] (page 8, line 281)

The “initial titer” of experiment of 56°C different from that of 40°C experiment because while propagating the virus strains in specific-pathogen-free (SPF) em- bryonated chicken eggs (ECE)  each batch/passage had different virus titres and we didnot adjusted to a common initial titre for experiment of 56°C and 40°C as the target was to assess the thermostability of the virus only irrespective of the titre. Rather, we have calculated standard dose of 10⁶ EID50 per chick for each virus strains used in the immunogenecity studies.

The rate constant K is determined using non-linear regression followed by exponential one-phase decay using GraphPad Prism ver. 9.3.0.
The rate constant and time constants are simply reciprocals of each other. Prism always fits the rate constant (k), but computes the time constant (tau) as well and reports the standard error and confidence interval of the time constant just as if the model had been written to fit that constant.
K is the rate constant, expressed in reciprocal of the X-axis time units. If X is in minutes, then K is expressed in inverse minutes.
* The half-life equals ln(2)/k where ln is the abbreviation for natural logarithm.

Throughout the manuscript, inferior letter should be used for number of EID50 and Log2.

We have made the changes.

 In line 362, Li et al [40] reported~.

We have made the change.